Thiosuccinyl-crosslinked Hemoglobin Analogs and Methods of Use and Preparation Thereof

ABSTRACT

Provided herein are thiosuccinyl-crosslinked hemoglobin analogs useful as blood replacement agents, pharmaceutical compositions comprising the same and the methods of use and preparation thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication No. 62/893,220 filed on Aug. 29, 2019, which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

The present disclosure generally relates to thiosuccinyl-crosslinkedhemoglobin analogs, pharmaceutical compositions comprising the same andthe methods of use and preparation thereof.

BACKGROUND

Hemoglobin is an iron-containing, oxygen-transport metalloproteinpresent in red blood cells of almost all vertebrates as well as thetissues of some invertebrates. Hemoglobin in red blood cells isresponsible for carrying oxygen from the lungs to the rest of the bodyand then returning carbon dioxide from the body cells to the lungs,where the carbon dioxide can be exhaled. Since hemoglobin has thisoxygen transport feature, purified hemoglobin from human- oranimal-derived red blood cells is an ideal material for use in thedevelopment of potent oxygen therapeutics, if it can be stabilized exvivo and used in vivo and ex vivo.

Naturally-occurring hemoglobin is a non-covalently linked heterotetramercomposed of 2α and 2β globin subunits, and it is generally stable whenpresent within red blood cells. However, when naturally-occurringhemoglobin is removed from red blood cells, it becomes unstable andsplits into 2 dimers (as globin chains) in blood circulation. Thedimeric hemoglobin is rapidly cleared from the kidney via glomerularfiltration, because of its lower molecular weight (approximately 32 kDa)(Bunn, H. F. et al., 1969, J Exp Med, 129: 909-23) or from the liver viathe haptoglobin-CD 163 pathway (Kristiansen, M. et al., 2001, Nature,409: 198-201). The breakdown of the tetramer linkage not only causesrenal injury when the dissociated hemoglobin is filtered through thekidneys and excreted, but also negatively impacts the sustainability ofthe functional hemoglobin in blood circulation.

To overcome these problems, researchers have attempted to developvarious types of modified hemoglobin (hemoglobin-based oxygen carrier,HBOC) with either a stable tetramer configuration or an increasedmolecular weight. These HBOCs are developed from hemoglobin purifiedfrom human- or animal-derived red blood cells and further modified byintra- or intermolecular cross-linking, polymerization, PEGylation orencapsulation (Keipert, P. E. et al., 1992, BiomaterArtif Cells ImmobilBiotechnol, 20: 737-45). Compared to stroma-free hemoglobin, these HBOCscan have longer blood retention and possess different oxygen transportcapacities based on the hemoglobin modification strategies used.

Although the safety, efficacy and pharmacokinetics of these HBOCs wereevaluated in preclinical and clinical trials (Jahr, J. S. et al., 2012,Curr Drug Discov Technol, 9: 158-65; Kim, H. W. & Greenburg, A. G, 2004,Artif Organs, 28: 813-28), the United States Food and DrugAdministration (FDA) has not approved any HBOCs for use in humans yet.Human hemoglobin products were developed since 1993 and evaluated inPhase III trials, but their development for clinical use was suspended,because of the undesirable side effects reported in clinical trialstudies (Hess, J. et al., 1991, Blood 78:356A; Jahr, J. S. et al., 2012,Curr Drug Discov Technol, 9: 158-65; Winslow, R. M., 2000, Vox Sang,79:1-20; Cheng, D. C. et al., 2004, J Thorac Cardiovasc Surg, 127:79-86; Greenburg, A. G. et al., 2004, J Am Coll Surg, 198: 373-383;Hill, S. E. et al., 2002, J Cardiothorac Vasc Anesth, 16: 695-702).

Although the failure of using acellular human HBOC products is reportedto be associated with a significantly increased risk of death andmyocardial infract compared to a control solution, a bovine hemoglobinglutamer-250, HBOC-201, from Hemopure, is approved for the treatment ofanemia and for use during surgery in South Africa since 2001 (Lok, C.,2001, Nature, 410:855; Mer, M. et al., 2016, Transfusion (Paris),56:2631-36) and it is also provided to patients with life-threateninganemia in the United States for whom allogeneic blood transfusion is notan option since 2014 (Lundy, J. B. et al., 2014, Int J Burns Trauma, 4:45-8; Posluszny, J. A. & Napolitano, L. M., 2016, Archives of TraumaResearch, 5: e30610; Resar, L. M. et al., 2016, Transfusion, 56:2637-47).

Even though renal toxicity issues are addressed by preventing hemoglobindissociation, vasoconstriction following the administration is anothersafety concern of HBOC products. It has been shown that acellularhemoglobin is much more effective in scavenging nitric oxide (NO) withrespect to hemoglobin inside red blood cells, which results invasoconstriction and hypertension observed with HBOC administration(Mozzarelli, A. et. al., 2010, Blood Transfus, 8(S3): s59-s68). The meanarterial blood pressure increased immediately after HBOC infusion andsuch increase is often associated with reduced cardiac output andincreased total peripheral resistance (Hess, J. R. het al., 1993, J ApplPhysiol, 74(4):1769-78). Vasoconstriction can limit tissue perfusion andoxygenation and is a severe adverse effect for patients that aresuffering from blood loss and hemodilution.

Although NO scavenging by HBOCs is considered the primary mechanismleading to vasoconstriction, other possible factors are considered, suchas oxygen pressure in the pre-capillary arterioles (Winslow, R. M.,2003, J Intern Med, 253(5): 508-17; Tsai, A. G. et al., 2003, Am JPhysiol Heart Circ Physiol, 284(4): H1411-9) and solution viscosity(Gaucher-Di, S. C. et al., 2009, Biomaterials, 30(4): 445-51). Inaddition to NO scavenging, other mechanisms of HBOC toxicity have alsobeen suggested, including oversupply of oxygen (Winslow, R. M., 2008,Biochim Biophys Acta, 2008, 1784: 1382-86) and heme-mediated oxidativeside reactions (Alayash, A. I., 1999, Nat Biotechnol, 17: 545-9;Alayash, A. I., 2004, Nat Rev Drug Discov, 3: 152-9).

Although some new HBOCs with less reactivity to NO and reduced abilityof oversupplying oxygen to guard against oxidative side reactions havebeen designed, some unexplained toxicities associated with these newlydesigned HBOCs were still observed in preclinical studies. These resultsrevealed that the diverse chemistry of HBOC modifications and its impacton HBOC toxicity are not fully understood and the side effect profilesof HBOCs vary depending on the nature of their chemical and/or geneticmodifications.

Recent developments in hemoglobin-based oxygen therapeutics haveincorporated various chemical approaches, such as polymerization,PEGylation, cross-linking and encapsulation, to form stabilizedmultimeric hemoglobin with improved side effect profiles, so that thestabilized hemoglobin can function outside the red blood cell. The priorart teaches that highly purified stroma-free hemoglobin can be obtainedby lysing animal red blood cells, followed by different purificationstrategies with heat treatment (Sakai, H. et al, 1994, Artif Cells BloodSubstit Immobil Biotechnol, 22(3):651-6), aqueous phase extraction (Lee,C. J. & Kan, P., 1993, U.S. Pat. No. 5,407,579), tangential flowfiltration (Palmer, A. F. et al, 2009, Biotechnol Prog, 25(6): 1803-9;Elmer, J. et al, 2009, Biotechnol Prog, 25(5): 1402-10) and/or anionexchange chromatography (Sun, G. & Palmer, A. F., 2008, J Chromatogr BAnalyt Technol Biomed Life Sci, 867(1):1-7; Houtchens, R. A. & Rausch,C. W., 2000, U.S. Pat. No. 6,150,507; Pliura, D. H. et al, 1996, U.S.Pat. No. 5,545,328). Highly purified, stroma-free hemoglobin can be usedto generate the hemoglobin-based product with designated oxygen affinityproperties through different chemical approaches. Among theseapproaches, chemical crosslinking is one common approach to formstabilized multimeric hemoglobin through intramolecular bonds within thetetramers as well as intermolecular bonds between the stabilizedtetramers. Such chemically stabilized hemoglobin can be further modifiedto form a HBOC with designated properties.

Bis-3,5-dibromosalicyl fumarate (DBSF), a chemical crosslinker used inhemoglobin processing, not only stabilizes hemoglobin throughintramolecular crosslinking, but also affects the oxygen affinity of thehemoglobin (Beanna, J. N. et al. U.S. Pat. No. 5,248,766; Wong B. L. etal. U.S. Pat. No. 8,106,011 B1). The stabilized multimeric hemoglobin isthe preferred form in order to reduce side effects of stroma-freehemoglobin and increase the circulatory half-life of the hemoglobin.However, the present inventors determined that the fumaryl moieties onthe crosslinked hemoglobin react with thiols in vitro and in vivo.Conventional hemoglobin products are commonly formulated with thethiol-containing excipients, such as N-acetyl cysteine (NAC) or cysteine(Cys), aiming to reduce the level of dysfunctional methemoglobin in thepharmaceutical composition (Beanna, J. N. et al. U.S. Pat. No.5,248,766; Timothy, E. E. U.S. Pat. No. 5,281,579; Wong B. L. et al.U.S. Pat. No. 8,106,011 B1). However, the reaction of thiol-containingexcipients with the fumaryl moieties of the crosslinked hemoglobinreduces their capability of reducing methemoglobin, which may affectHBOC product stability during product storage.

In humans, the redox status in plasma is tightly regulated by lowmolecular weight thiols, such as glutathione, cysteine (Cys) andhomocysteine. An imbalance in plasma redox status is found to beassociated with cardiovascular disease (Brunelli, E et al. Oxid Med CellLongev, 1-11, 2017). Therefore, the stabilized multimeric hemoglobinwith crosslinkers containing fumaryl group taught by the prior art cancause in-vitro instability of the pharmaceutical composition and in vivoredox system imbalance, is a problem. There is thus a need in the artfor a technique to create stabilized multimeric hemoglobin not only withan improved product safety profile and desirable oxygen-carryingproperties, but also containing crosslinkers that do not react withthiols in-vitro and in-vivo. Such stabilized and crosslinked hemoglobincan be used in a wide variety of medical applications, depending upondifferent medical applications; where different levels of oxygenaffinity are desirable.

SUMMARY

The present disclosure generally relates to thiosuccinyl-crosslinkedhemoglobin, pharmaceutical compositions comprising the same, and methodsof use and preparation thereof. At least one of the thiosuccinylcrosslinkers described herein are useful for hemoglobin stabilization. Aschematic diagram of the formation of thiosuccinyl-crosslinkedhemoglobin is depicted in FIG. 1. The thiosuccinyl-crosslinkedhemoglobin can be produced by intermolecular 1,4-addition of thiol tohemoglobin β-β crosslinked with at least one fumaryl crosslinkers.

Provided herein is a thiosuccinyl-crosslinked hemoglobin that overcomesthe stability issues associated with previously known stabilizedhemoglobin with crosslinkers containing a fumaryl group, by avoiding thereaction between the stabilized hemoglobin and thiols in-vitro andin-vivo. Surprisingly, conjugation of thiols to the fumaryl moieties ofthe crosslinked hemoglobin also improves the in-vivo tissue oxygenation,but advantageously does not alter its p50 value.

In a first aspect, provided herein is a thiosuccinyl-crosslinkedhemoglobin comprising a tetrameric hemoglobin and at least onethiosuccinyl crosslinking moiety of Formula 1:

or a pharmaceutically acceptable salt or zwitterion thereof, wherein

each N* independently represents a nitrogen selected from the groupconsisting of a nitrogen in a lysine residue side chain in thetetrameric hemoglobin and a nitrogen at a N-terminus in the tetramerichemoglobin; and

R¹ is alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl,heteroaryl, or —(CR₂)_(n)Y, wherein n is an integer selected from 0-10;R for each instance is independently hydrogen, alkyl, aralkyl, alkenyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; or two instances of Rtaken together form a 3-6 membered cycloalkyl or heterocycloalkylcontaining 1, 2, or 3 heteroatoms selected from N, O, and S; and Y isselected from the group consisting of OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴,—(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, —(NR⁴)S(O)₂OR⁴, and —(CRR²R³), wherein R² ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴,—O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; R³ ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴,—O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; and R⁴for each instance is independently selected from the group consisting ofhydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; or R¹ is a moiety selected from the group consisting of:

and

N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.

In certain embodiments, R¹ is a moiety of Formula 2:

wherein n is a whole number selected from the group consisting of 0, 1,2, 3, and 4;

R for each instance is independently selected from the group consistingof hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl;

R² is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —N(R⁴)₂, —NH(C═O)R⁴, or —NH(C═O)N(R⁴)₂;

R³ is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —CO₂R⁴, —(C═O)NHR⁴, —OR⁴, or —N(R⁴)₂; and

R⁴ for each instance is independently selected from the group consistingof hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl;

or R¹ is a moiety selected from the group consisting of:

and

N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.

In certain embodiments, n is 1 or 2; R is hydrogen; R² is —NHR⁴,—NH(C═O)R⁴, or —NH(C═O)R⁴N(R⁴)₂; and R³ is hydrogen, —OR⁴, —CO₂R⁴, or—(C═O)NHR⁴, wherein R⁴ for each instance is independently selected fromthe group consisting of hydrogen and alkyl.

In certain embodiments, R¹ is selected from the group consisting of:

or a pharmaceutically acceptable salt or zwitterion thereof, wherein mis a whole number selected from 1-1000.

In certain embodiments, each N* independently represents a nitrogenselected from the group consisting of a nitrogen in a lysine residueside chain in a beta globin chain of the tetrameric hemoglobin and anitrogen at a N-terminus in a beta globin chain of the tetramerichemoglobin.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobin issubstantially pure.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobincomprises 1, 2, or 3 thiosuccinyl crosslinking moiety of Formula 1.

In certain embodiments, the at least one thiosuccinyl crosslinkingmoiety crosslinks two beta globin chains of the tetrameric hemoglobin.

In certain embodiments, the tetrameric hemoglobin is human hemoglobin,bovine hemoglobin, or porcine hemoglobin.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobin issubstantially stroma-free.

In a second aspect, provided herein is a pharmaceutical compositioncomprising at least one of the thiosuccinyl-crosslinked hemoglobin asdescribed herein and at least one pharmaceutically acceptable excipient.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobin ispresent in the pharmaceutical composition at a weight percentage between10-90%.

In certain embodiments, the pharmaceutical composition comprisesthiosuccinyl-crosslinked hemoglobin comprising 1, 2, or 3 thiosuccinylcrosslinking moieties of Formula 1; or a combination thereof.

In a third aspect, provided herein is a method for preparing thethiosuccinyl-crosslinked hemoglobin as described herein comprising thesteps of:

contacting a tetrameric hemoglobin with a fumaryl crosslinking agentthereby forming a fumaryl-crosslinked hemoglobin; contacting thefumaryl-crosslinked hemoglobin with a thiol or a pharmaceuticallyacceptable salt or zwitterion thereof thereby forming thethiosuccinyl-crosslinked hemoglobin as described herein.

In certain embodiments, the fumaryl crosslinking agent is selected fromthe group consisting of bis-3,5-dibromosalicyl fumarate (DBSF), fumarylchloride and bis(salicyl) fumarate.

In certain embodiments, the thiol has the formula: R¹SH or apharmaceutically acceptable salt or zwitterion thereof, wherein R¹ isalkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl, heteroaryl,or —(CR₂)_(n)Y, wherein n is an integer selected from 0-10; R for eachinstance is independently hydrogen, alkyl, aralkyl, alkenyl, cycloalkyl,heterocycloalkyl, aryl, or heteroaryl; or two instances of R takentogether form a 3-6 membered cycloalkyl or heterocycloalkyl containing1, 2, or 3 heteroatoms selected from N, O, and S; and Y is selected fromthe group consisting of R¹ is alkyl, alkenyl, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or —(CR₂)_(n)Y, wherein nis an integer selected from 0-10; R for each instance is independentlyhydrogen, alkyl, aralkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl,or heteroaryl; or two instances of R taken together form a 3-6 memberedcycloalkyl or heterocycloalkyl containing 1, 2, or 3 heteroatomsselected from N, O, and S; and Y is selected from the group consistingof OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴,—(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, —(NR⁴)S(O)₂OR⁴, and—(CRR²R³), wherein R² is hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴,—(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; R³ is hydrogen, alkyl, aralkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR⁴, SR⁴, N(R⁴)₂,—(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; and R⁴ for each instance isindependently selected from the group consisting of hydrogen, alkyl,aralkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; or R¹ is amoiety selected from the group consisting of:

and

N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.

In certain embodiments, the thiol has the Formula 3:

or a pharmaceutically acceptable salt or zwitterion thereof, wherein

n is an integer selected from the group consisting of 0, 1, 2, 3, and 4;

R for each instance is independently selected from the group consistingof hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl;

R² is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —N(R⁴)₂, or —NH(C═O)R⁴;

R³ is hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —CO₂R⁴, —(C═O)NHR⁴, —OR⁴, or —N(R⁴)₂; and

R⁴ for each instance is independently selected from the group consistingof hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; or the thiol is selected from the group consisting ofdithiothreitol, HS(CH₂CH₂O)_(m)CH₃, HS(CH₂CH₂O)_(m)H, glutathione or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected between 1-1000.

In certain embodiments, n is 1 or 2; R is hydrogen; R² is —NHR⁴,—NH(C═O)R⁴, or —NH(C═O)(NR⁴)₂; and R³ is hydrogen, —OR⁴, —CO₂R⁴, or—(C═O)NHR⁴, wherein R⁴ for each instance is independently selected fromthe group consisting of hydrogen and alkyl.

In certain embodiments, the thiol is selected from the group consistingof:

dithiothreitol, HS(CH₂CH₂O)_(m)CH₃, and HS(CH₂CH₂O)_(m)H or apharmaceutically acceptable salt or zwitterion thereof, wherein m is awhole number selected between 1-1000.

In certain embodiments, the step of contacting the fumaryl-crosslinkedhemoglobin with a thiol or a pharmaceutically acceptable salt orzwitterion thereof, the fumaryl-crosslinked hemoglobin and the thiol arepresent in a molar ratio of at least 1:1; 1:2; or 1:3.

In certain embodiments, the fumaryl-crosslinked hemoglobin and the thiolare present in a molar ratio of greater than 1:3.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobin is inisolated and substantially pure form.

In a fourth aspect, provided herein is a method for increasing thevolume of the blood circulatory system in a subject in need thereof,wherein the method comprises transfusing into the system of the subjecta therapeutically effective amount of the thiosuccinyl-crosslinkedhemoglobin described herein

In a fifth aspect, provided herein is a method for the treatment ofshock in a subject in need thereof, wherein the method comprisestransfusing into the system of the subject a therapeutically effectiveamount of the thiosuccinyl-crosslinked hemoglobin described herein.

In a sixth aspect, provided herein is a method of supplying oxygen tothe tissues and organs in a subject in need thereof, wherein the methodcomprises transfusing into the system of the subject a therapeuticallyeffective amount of the thiosuccinyl-crosslinked hemoglobin describedherein.

In a seventh aspect, provided herein is a method of treating cancer in asubject in need thereof, wherein the method comprises transfusing intothe system of the subject a therapeutically effective amount of thethiosuccinyl-crosslinked hemoglobin described herein, wherein the canceris triple-negative breast cancer or colorectal cancer.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobin issubstantially pure.

The present disclosure relates to the modification of thefumaryl-crosslinked hemoglobin by thiols. In certain embodiments, themodification of the fumaryl-crosslinked hemoglobin by thiols alters thep50 value of the fumaryl-crosslinked hemoglobin no greater than 10%. Inthe examples below, the infusion of 650 mg/kg cysteinyl-succinylcrosslinked hemoglobin, which is produced by the conjugation of cysteineto the fumaryl moieties of the fumaryl-crosslinked hemoglobin, shows asignificant increase in liver tissue oxygenation in a shock rat model.Moreover, its efficacy enhancement is further demonstrated by theimproved restoration of blood flow in a peripheral artery disease micemodel, compared to fumaryl-crosslinked hemoglobin throughout theexperiment.

The present disclosure also provides a method of making thethiosuccinyl-crosslinked hemoglobin. In certain embodiments, the methodcomprises the steps of 1) reducing the dissolved oxygen level of thesolution containing the fumaryl-crosslinked hemoglobin down to 0.1 mg/L;2) mixing the fumaryl-crosslinked hemoglobin solution with thiol underlow oxygen conditions to form the thiosuccinyl-crosslinked hemoglobinunder a condition in which at least 95% of the fumaryl bridges reactwith the thiol reagent as well as the methemoglobin level of thethiosuccinyl-crosslinked hemoglobin is reduced to less than 2% and 3)optionally removing any residual thiols to less than 0.03% (w/w).N-acetyl cysteine (NAC) is added at a concentration of approximately0.05-0.2% to further reduce the methemoglobin produced from theproduction process and also prevent its formation during storage. Thepresent disclosure further provides a method to prepare thethiosuccinyl-crosslinked hemoglobin in high yield and purity by, e.g.,adjusting the equivalence of the thiol reagent used in the couplingreaction, pH value and duration of the reaction.

The method of the present disclosure can be used to preparethiosuccinyl-crosslinked hemoglobin having a p50 ranging from about 5-70mmHg as measured at 37° C. and pH 7.4. Different levels of oxygenaffinity are desirable, depending upon the intended chemicalmodification of hemoglobin and medical application. In theseembodiments, the thiol-containing reagents used for conjugating to thefumaryl moieties of crosslinked hemoglobin can include, for example,cysteine, NAC, β-mercaptoethanol, and other thiol compounds as describedherein. The thiosuccinyl-crosslinked hemoglobin can be a tetramerichemoglobin with a molecular weight of about 65 kDa containing at leastone (1XL), two (2XL) or three (3XL) alkyl thiol crosslinker(s) between βglobin chains.

In the examples below, a solution containing cysteinyl-succinylcrosslinked bovine hemoglobin is produced by conjugating cysteine to thefumaryl moieties of fumaryl-crosslinked bovine hemoglobin. In themodification step, 40-80 mM cysteine at pH 8.0-8.3 is incubated with thetetrameric hemoglobin (tHb=7-10 g/dL) for a period of 15-30 hours at10-30° C. under deoxygenated conditions (e.g., dissolved oxygen (DO)levels maintained below 0.1 mg/L). Up to 95% modification of the fumarylmoieties in the fumaryl-crosslinked hemoglobin by cysteine can beachieved and the methemoglobin level of the cysteinyl-succinylcrosslinked hemoglobin can be reduced to less than 2% aftermodification. The residual cysteine/cystine in the reaction mixture canbe removed by a filtration step using a 30 kDa NMWCO membrane throughwhich the reaction mixture can be filtered through 10-16 diafiltrationvolume (DV) with acetate buffer (99 mM NaCl, 46 mM sodium acetate) tobring the cysteine and cystine levels below 0.03% (w/w).

The pharmaceutical composition comprising cysteinyl-succinyl crosslinkedbovine hemoglobin can be kept under nitrogen with the presence of 0.2%(w/w) NAC with the following product characteristics: tHb=9.5-10.5 g/dL,pH 7.4-8.4, O₂Hb≤10%, MetHb (methemoglobin) ≤5%, endotoxin ≤0.25 EU/mLand cysteinyl-succinyl crosslinked hemoglobin in range of 95-100%purity.

The present disclosure also provides a pharmaceutical compositioncomprising the thiosuccinyl-crosslinked hemoglobin, such as thecysteinyl-succinyl crosslinked hemoglobin. Such compositions can be usedfor improving the delivery of oxygen and treatment against global andregional ischemic/hypoxic conditions, including hemorrhagic shock,myocardial ischemia reperfusion injury, peripheral artery disease andtraumatic brain injury. In addition, such composition is also used fortreating autoimmune diseases and cancer treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present disclosure willbecome apparent from the following description of the disclosure, whentaken in conjunction with the accompanying drawings.

FIG. 1 is a schematic depiction of the formation ofthiosuccinyl-crosslinked hemoglobin.

FIG. 2 is a flow-chart depicting the method of formation ofcysteinyl-succinyl crosslinked hemoglobin.

FIG. 3 depicts the size exclusion chromatogram for (A) non-crosslinkedbovine hemoglobin and (B) fumaryl-crosslinked hemoglobin by DBSFreaction.

FIG. 4 depicts deconvoluted ESI-MS spectrum of (A) fumaryl-crosslinkedhemoglobin by DBSF reaction and (B) cysteinyl-succinyl crosslinkedhemoglobin.

FIG. 5 depicts ESI-MS/MS spectrum of cysteinyl-succinyl peptide,demonstrating confirmation of cysteine modification on the double bondof fumaryl group in cysteinyl-succinyl crosslinked hemoglobin.

FIG. 6 depicts deconvoluted ESI-MS spectra of the reactivity of thedouble bond of fumaryl group towards different thiol-containing reagents(cysteine, β-mercaptoethanol and homocysteine).

FIG. 7 depicts deconvoluted ESI-MS spectra of the reactivity of ahemoglobin crosslinked with bis-3,5-dibromosalicyl succinate (DBSS)towards cysteine and N-acetyl cysteine (NAC) at Day 2 and 9.

FIG. 8 represents conversion scheme of fumaramide (a) BocLysF withβ-mercaptoethanol to give the alkylthiosuccinamide, (b) BocLysF-BME andwith cysteine to give the alkylthiosuccinamide (c) BocLysF-Cys.

FIG. 9 depicts the stability of NAC and NAC₂ incysteinyl-succinyl-crosslinked hemoglobin and fumaryl-crosslinkedhemoglobin solution, respectively.

FIG. 10 depicts deconvoluted ESI-MS spectrum of (A) cysteinyl-succinylcrosslinked hemoglobin and (B) fumaryl-crosslinked hemoglobin,demonstrating in vivo stability of the hemoglobin in the bloodcirculation.

FIG. 11 shows the injection of 650 mg/kg cysteinyl-succinyl crosslinkedhemoglobin solution results in a significant increase in liver tissueoxygen level comparing with fumaryl-crosslinked hemoglobin solution inrat severe hemorrhagic shock. Data are reported as mean±SD. Statisticalanalysis is performed by two-way ANOVA. ***p<0.001 vsfumaryl-crosslinked hemoglobin solution.

FIG. 12 shows an increase in Oxy-Hb level in the ischemic limb followingcysteinyl-succinyl crosslinked hemoglobin and fumaryl-crosslinkedhemoglobin treatment. Oxy-Hb is measured by the TiVi 700 TissueViability Imager with Oxygen Mapper before and after inducing limbischemia, and at different time points up to 21 days followingtreatment. For cysteinyl-succinyl crosslinked hemoglobin treatment, asignificant increase in Oxy-Hb is observed in 30 minutes post infusion,and up to 21 days post treatment whereas fumaryl-crosslinked hemoglobinshows a significant increase in 14 days post treatment and 21 days posttreatment. The Oxy-Hb level is expressed as the flux density measured inthe ligated limb divided by the control limb (without ligation) in thesame mouse at the same time point. Data are reported as mean±SD.*p<0.05, **p<0.01 vs control group.

FIG. 13 shows a restoration of perfusion in ischemic limb followingcysteinyl-succinyl crosslinked hemoglobin and fumaryl-crosslinkedhemoglobin treatment. Blood perfusion is measured by the Moor SerialLaser Doppler Imager before and after inducing limb ischemia, and atdifferent time points up to 21 days following treatment. A significantrestoration of perfusion is observed up to 21 days followingcysteinyl-succinyl crosslinked hemoglobin and fumaryl-crosslinkedhemoglobin treatment. Data are reported as mean±SD. *p<0.05, **p<0.01,***p<0.001 vs control group.

FIG. 14 shows maintenance of mesenchymal stem cell (MSC) populations inlimb ischemia following cysteinyl-succinyl crosslinked hemoglobin andfumaryl-crosslinked hemoglobin treatment. An increase in circulatingCD45⁻CD29⁺, CD45⁻CD105⁺, CD45⁻CD106⁺ MSC populations is observed afterinducing limb ischemia and which sustains for a longer period of timecompared with control. A more significant improvement in MSC populationsis observed upon cysteinyl-succinyl crosslinked hemoglobin treatment.Data are reported as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs controlgroup.

FIG. 15 shows a cysteinyl-succinyl crosslinked hemoglobin restoredoxygenation in muscle tissue (triceps) under a non-human primate(cynomolgus monkey) model of severe hemorrhagic shock. Infusion of 500mg/kg cysteinyl-succinyl crosslinked hemoglobin results in an increasein muscle tissue oxygenation when comparing with autologous plasmatreatment (control). Data are reported as mean±SD.

FIG. 16 shows a restoration of mean arterial pressure in a non-humanprimate model of severe hemorrhagic shock following cysteinyl-succinylcrosslinked hemoglobin treatment. Comparing with autologous plasmatreatment, a higher mean arterial pressure is maintained up to 3 hoursfollowing the cysteinyl-succinyl crosslinked hemoglobin treatment. Dataare reported as mean±SD.

FIG. 17 shows a dose-dependent increase in Oxy-Hb level in the ischemiclimb following treatment with cysteinyl-succinyl crosslinked hemoglobinsolution. Oxy-Hb is measured by the TiVi 700 Tissue Viability Imagerwith Oxygen Mapper before and after inducing limb ischemia, and atdifferent time points up to 21 days following treatment withcysteinyl-succinyl crosslinked hemoglobin solution (n=8 per group). Asignificant and dose-dependent increase in Oxy-Hb is observed in 1 hourpost infusion, and up to 21 days post treatment. The Oxy-Hb level isexpressed as the flux density measured in the ligated limb divided bythe control limb (without ligation) in the same mouse at the same timepoint. Data are reported as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vsnegative control group.

FIG. 18 shows a dose-dependent restoration of perfusion in ischemic limbfollowing treatment with cysteinyl-succinyl crosslinked hemoglobinsolution. Blood perfusion is measured by the Moor Serial Laser DopplerImager before and after inducing limb ischemia, and at different timepoints up to 21 days following treatment with cysteinyl-succinylcrosslinked hemoglobin solution. A significant and dose-dependentrestoration of perfusion is observed up to 21 days following treatmentwith cysteinyl-succinyl crosslinked hemoglobin solution (n=8 per group).Data are reported as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs negativecontrol group.

FIG. 19 depicts a selective activation of mesenchymal stem cell (MSC)populations in limb ischemia following treatment with cysteinyl-succinylcrosslinked hemoglobin solution. A significant and dose-dependentincrease in circulating CD45⁻CD29⁺, CD45⁻CD105⁺, CD45⁻CD106⁺ MSCpopulations is observed after inducing limb ischemia and which sustainedfor a longer period of time compared with negative control. Data arereported as mean±SD. *p<0.05, **p<0.01, ***p<0.001 vs negative controlgroup.

FIG. 20 shows a significant reduction of myocardial infraction areafollowing treatment with cysteinyl-succinyl crosslinked hemoglobinsolution. Histological analysis revealed a comparable area at risk isinduced between the control and treatment groups following LAD ligation(Left graph). A significant reduction in the infarct area is observed inthe groups receiving treatment with 100 mg/kg cysteinyl-succinylcrosslinked hemoglobin solution (Right graph).

FIG. 21 is a schematic depiction of lupus induction and treatment in aC57 mouse model. Lupus nephritis is induced by bi-weekly injection ofapoptotic cell nuclear extracts with Freud's adjuvant for a total of 3times while treatment (800 mg/kg cysteinyl-succinyl crosslinkedhemoglobin) is given twice per week for 2 weeks. Disease progression iscontinued for 12 weeks from the first induction for pathologicalanalysis.

FIG. 22 shows a decreased immunoglobulin (Ig, score: 0-3) isotype M(IgM) and isotype G (IgG) deposition on glomerulus in kidneys. *p<0.05,***p<0.001.

FIG. 23 shows lower glomerular activity index with significantly reducedcellular proliferation, leukocyte infiltration, fibrinoidnecrosis/karyorrhexis, cellular crescents and hyaline thrombi, wireloops in the kidneys. *p<0.05, **p<0.01, ***p<0.001.

FIG. 24 shows an acute treatment with cysteinyl-succinyl crosslinkedhemoglobin improved neurological score following TBI. The compositeneuroscore is obtained using a battery of 8 neurological assessments at4 hour, 1 day, 3 days and 7 days following TBI. Graph depicts effect oftreatment with cysteinyl-succinyl crosslinked hemoglobin solution onneuroscore at different time points. Cysteinyl-succinyl crosslinkedhemoglobin-treated TBI animals show significant increase in neuroscore24 hours after the treatment (*p<0.05). Two-way repeated measures ANOVAfollowed by Sidak post hoc-test; data expressed as Mean±SEM.

FIG. 25 shows an acute treatment with cysteinyl-succinyl crosslinkedhemoglobin improved motor recovery following TBI. Efficacy of treatmentwith cysteinyl-succinyl crosslinked hemoglobin solution on motorfunctions at 1 h post-TBI is assessed using the horizontal ladder taskand the cylinder test. The left graph represents the effect of treatmentwith cysteinyl-succinyl crosslinked hemoglobin on error score on theladder test. Cysteinyl-succinyl crosslinked hemoglobin significantlyimproves the ladder test performance in TBI rats as compared to thecontrol group on the Day 3 (*p<0.05) but not on Day 7. Only the errorscore of the control group on Day 7 is significantly lower than that onDay 3 (**p<0.01) while the treatment group reaches the same recoverylevel at Day 3. The right graph shows the degree of asymmetrical paw useduring cylinder test conducted on Day 3 and Day 7 following TBI. Nosignificant difference in the use of affected paw between control groupand treatment group (p>0.05) has been found. Data expressed as Mean±SEM.

FIG. 26 shows the reduction of TBI-induced astrogliosis by treatmentwith cysteinyl-succinyl crosslinked hemoglobin solution. Effect of asingle acute treatment with cysteinyl-succinyl crosslinked hemoglobinsolution on astrocyte activation is assessed with GFAPimmunofluorescence performed in brain tissue 7 days post-TBI. (A) Graphshows mean cell area of GFAP-positive astrocytes in both ipsilateral(Ipsi) and contralateral (Contra) hippocampus and thalamus. (*p<0.05),two-tailed t test; data expressed as Mean±SEM. (B) Representative 200×magnified images after background threshold using ImageJ of activatedastrocytes from ipsilateral hippocampus (dental gyrus and CA region),ipsilateral thalamus and, contralateral hippocampus (CA region) for bothcontrol group (upper panel) and treatment group (lower panel). DG:Dentate gyrus, CA: Hippocampal CA region, ThaI: Thalamus, i:Ipsilateral, c: Contralateral with respect to injury site.

DETAILED DESCRIPTION Definitions

The following terms shall be used to describe the present invention. Inthe absence of a specific definition set forth herein, the terms used todescribe the present invention shall be given their common meaning asunderstood by those of ordinary skill in the art.

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings can alsoconsist essentially of, or consist of, the recited components, and thatthe processes of the present teachings can also consist essentially of,or consist of, the recited process steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components. Further, it should be understood that elements and/orfeatures of a composition or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

As used herein, the terms “treat”, “treating”, “treatment”, and the likerefer to reducing or ameliorating a disorder/disease and/or symptomsassociated therewith. It will be appreciated, although not precluded,treating a disorder or condition does not require that the disorder,condition, or symptoms associated therewith be completely eliminated. Incertain embodiments, treatment includes prevention of a disorder orcondition, and/or symptoms associated therewith. The term “prevention”or “prevent” as used herein refers to any action that inhibits or atleast delays the development of a disorder, condition, or symptomsassociated therewith. Prevention can include primary, secondary andtertiary prevention levels, wherein: a) primary prevention avoids thedevelopment of a disease; b) secondary prevention activities are aimedat early disease treatment, thereby increasing opportunities forinterventions to prevent progression of the disease and emergence ofsymptoms; and c) tertiary prevention reduces the negative impact of analready established disease by restoring function and reducingdisease-related complications.

The term “subject” as used herein, refers to an animal, typically amammal or a human, that will be or has been the object of treatment,observation, and/or experiment. When the term is used in conjunctionwith administration of a compound described herein, then the subject hasbeen the object of treatment, observation, and/or administration of thecompound described herein.

The term “therapeutically effective amount” as used herein, means thatamount of the compound or pharmaceutical agent that elicits a biologicaland/or medicinal response in a cell culture, tissue system, subject,animal, or human that is being sought by a researcher, veterinarian,clinician, or physician, which includes alleviation of the symptoms ofthe disease, condition, or disorder being treated.

The term “composition” is intended to encompass a product comprising thespecified ingredients in the specified amounts, as well as any productthat results, directly or indirectly, from combinations of the specifiedingredients in the specified amounts.

The term “pharmaceutically acceptable carrier” refers to a medium thatis used to prepare a desired dosage form of a compound. Apharmaceutically acceptable carrier can include one or more solvents,diluents, or other liquid vehicles; dispersion or suspension aids;surface active agents; isotonic agents; thickening or emulsifyingagents; preservatives; solid binders; lubricants; and the like.Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1975) and Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe ed. (American PharmaceuticalAssoc. 2000), disclose various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof.

As used herein, unless otherwise indicated, the term “halo” or “halide”includes fluoro, chloro, bromo or iodo.

As used herein, “alkyl” refers to a straight-chain or branched saturatedhydrocarbon group. Examples of alkyl groups include methyl-, ethyl-,propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, iso-butyl,sec-butyl, tert-butyl), pentyl groups (e.g., 1-methylbutyl,2-methylbutyl, iso-pentyl, tert-pentyl, 1,2-dimethylpropyl, neopentyl,and 1-ethylpropyl), hexyl groups, and the like. In various embodiments,an alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl group),for example, 1-30 carbon atoms (i.e., C1-30 alkyl group). In certainembodiments, an alkyl group can have 1 to 6 carbon atoms, and can bereferred to as a “lower alkyl group.” Examples of lower alkyl groupsinclude methyl, ethyl, propyl (e.g., n-propyl and isopropyl), and butylgroups (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl). In certainembodiments, alkyl groups can be optionally substituted as describedherein. An alkyl group is generally not substituted with another alkylgroup, an alkenyl group, or an alkynyl group.

As used herein, “alkenyl” refers to a straight-chain or branched alkylgroup having one or more carbon-carbon double bonds. Examples of alkenylgroups include ethenyl, propenyl, butenyl, pentenyl, hexenyl,butadienyl, pentadienyl, hexadienyl groups, and the like. The one ormore carbon-carbon double bonds can be internal (such as in 2-butene) orterminal (such as in 1-butene). In various embodiments, an alkenyl groupcan have 2 to 40 carbon atoms (i.e., C2-40 alkenyl group), for example,2 to 20 carbon atoms (i.e., C2-20 alkenyl group). In certainembodiments, alkenyl groups can be substituted as described herein. Analkenyl group is generally not substituted with another alkenyl group,an alkyl group, or an alkynyl group.

As used herein, “cycloalkyl” by itself or as part of another substituentmeans, unless otherwise stated, a monocyclic hydrocarbon having between3-12 carbon atoms in the ring system and includes hydrogen, straightchain, branched chain, and/or cyclic substituents. Exemplary cycloalkylsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and the like.

As used herein, a “fused ring” or a “fused ring moiety” refers to apolycyclic ring system having at least two rings where at least one ofthe rings is aromatic and such aromatic ring (carbocyclic orheterocyclic) has a bond in common with at least one other ring that canbe aromatic or non-aromatic, and carbocyclic or heterocyclic. Thesepolycyclic ring systems can be highly p-conjugated and optionallysubstituted as described herein.

As used herein, “heteroatom” refers to an atom of any element other thancarbon or hydrogen and includes, for example, nitrogen, oxygen, silicon,sulfur, phosphorus, and selenium.

As used herein, “aryl” refers to an aromatic monocyclic hydrocarbon ringsystem or a polycyclic ring system in which two or more aromatichydrocarbon rings are fused (i.e., having a bond in common with)together or at least one aromatic monocyclic hydrocarbon ring is fusedto one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl groupcan have 6 to 24 carbon atoms in its ring system (e.g., C6-24 arylgroup), which can include multiple fused rings. In certain embodiments,a polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ringposition of the aryl group can be covalently linked to the definedchemical structure. Examples of aryl groups having only aromaticcarbocyclic ring(s) include phenyl, 1-naphthyl (bicyclic), 2-naphthyl(bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic),pentacenyl (pentacyclic), and like groups. Examples of polycyclic ringsystems in which at least one aromatic carbocyclic ring is fused to oneor more cycloalkyl and/or cycloheteroalkyl rings include, among others,benzo derivatives of cyclopentane (i.e., an indanyl group, which is a5,6-bicyclic cycloalkyl/aromatic ring system), cyclohexane (i.e., atetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl/aromaticring system), imidazoline (i.e., a benzimidazolinyl group, which is a5,6-bicyclic cycloheteroalkyl/aromatic ring system), and pyran (i.e., achromenyl group, which is a 6,6-bicyclic cycloheteroalkyl/aromatic ringsystem). Other examples of aryl groups include benzodioxanyl,benzodioxolyl, chromanyl, indolinyl groups, and the like. In certainembodiments, aryl groups can be optionally substituted. In certainembodiments, an aryl group can have one or more halogen substituents,and can be referred to as a “haloaryl” group. Perhaloaryl groups, i.e.,aryl groups where all of the hydrogen atoms are replaced with halogenatoms (e.g., —C₆F₅), are included within the definition of “haloaryl.”In certain embodiments, an aryl group is substituted with another arylgroup and can be referred to as a biaryl group. Each of the aryl groupsin the biaryl group can be optionally substituted.

The term “aralkyl” refers to an alkyl group substituted with an arylgroup.

As used herein, “heteroaryl” refers to an aromatic monocyclic ringsystem containing at least one ring heteroatom selected from oxygen (O),nitrogen (N), sulfur (S), silicon (Si), and selenium (Se) or apolycyclic ring system where at least one of the rings present in thering system is aromatic and contains at least one ring heteroatom.Polycyclic heteroaryl groups include those having two or more heteroarylrings fused together, as well as those having at least one monocyclicheteroaryl ring fused to one or more aromatic carbocyclic rings,non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkylrings. A heteroaryl group, as a whole, can have, for example, 5 to 24ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 memberedheteroaryl group). The heteroaryl group can be attached to the definedchemical structure at any heteroatom or carbon atom that results in astable structure. Generally, heteroaryl rings do not contain O—O, S—S,or S—O bonds. However, one or more N or S atoms in a heteroaryl groupcan be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiopheneS,S-dioxide). Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl),SiH₂, SiH(alkyl), Si(alkyl)₂, SiH(arylalkyl), Si(arylalkyl)₂, orSi(alkyl)(arylalkyl). Examples of such heteroaryl rings includepyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl,thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl,benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl,quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl,benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl,cinnolinyl, IH-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl,naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl,thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl,pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl,thienoxazolyl, thienoimidazolyl groups, and the like. Further examplesof heteroaryl groups include 4,5,6,7-tetrahydroindolyl,tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups,and the like. In certain embodiments, heteroaryl groups can besubstituted as described herein. In certain embodiments, heteroarylgroups can be optionally substituted.

The term “optionally substituted” refers to a chemical group, such asalkyl, cycloalkyl aryl, and the like, wherein one or more hydrogen maybe replaced with a substituent as described herein, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like

The term “carbocycle” is art-recognized and refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “nitro” is art-recognized and refers to —NO₂; the term“halogen” is art-recognized and refers to —F, —Cl, —Br or —I; the term“sulfhydryl” is art-recognized and refers to —SH; the term “hydroxyl”means —OH; and the term “sulfonyl” and “sulfone” is art-recognized andrefers to —SO₂—. “Halide” designates the corresponding anion of thehalogens.

The term “alkylthio” refers to a hydrocarbyl group having a sulfurradical attached thereto. In certain embodiments, the “alkylthio” moietyis represented by one of —S-alkyl, —S-alkenyl, or —S-alkynyl.Representative alkylthio groups include methylthio, ethylthio, and thelike.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of subjects without unduetoxicity, irritation, allergic response and the like, and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.describes pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptablesalts of the compounds provided herein include those derived fromsuitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, besylate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. In certain embodiments, organic acids from which salts canbe derived include, for example, acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinicacid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamicacid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like.

Pharmaceutically acceptable salts derived from appropriate bases includealkali metal, alkaline earth metal, ammonium and N*(C₁₋₄alkyl)₄ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, iron, zinc, copper, manganese,aluminum, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, non-toxic ammonium, quaternary ammonium, andamine cations formed using counterions, such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, andaryl sulfonate. Organic bases from which salts can be derived include,for example, primary, secondary, and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines, basicion exchange resins, and the like, such as isopropylamine,trimethylamine, diethylamine, triethylamine, tripropylamine, andethanolamine. In certain embodiments, the pharmaceutically acceptablebase addition salt is chosen from ammonium, potassium, sodium, calcium,and magnesium salts.

As used herein, the term “isolated” in connection with a compounddescribed herein means the compound is not in a cell or organism and thecompound is separated from some or all of the components that typicallyaccompany it in a cell or organism.

As used herein, the term “substantially pure” in connection with asample of a compound described herein means the sample contains at least60% by weight of the compound. In certain embodiments, the samplecontains at least 70% by weight of the compound; at least 75% by weightof the compound; at least 80% by weight of the compound; at least 85% byweight of the compound; at least 90% by weight of the compound; at least95% by weight of the compound; or at least 98% by weight of thecompound.

As used herein, the term “substantially stroma-free” in connection witha sample of a compound described herein means the sample contains lessthan 5% by weight stroma. In certain embodiments, the samples containsless than 4% by weight stroma; less than 3% by weight stroma; less than2% by weight stroma; less than 1% by weight stroma; less than 0.5% byweight stroma; less than 0.1% by weight stroma; less than 0.05% byweight stroma; or less than 0.01% by weight stroma.

The present disclosure provides a thiosuccinyl-crosslinked hemoglobincomprising a tetrameric hemoglobin and at least one thiosuccinylcrosslinking moiety of Formula 1:

or a pharmaceutically acceptable salt or zwitterion thereof, whereineach N* independently represents a nitrogen selected from the groupconsisting of a nitrogen in a lysine residue side chain in thetetrameric hemoglobin and a nitrogen at a N-terminus in the tetramerichemoglobin; and

R¹ is alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl,heteroaryl, or —(CR₂)_(n)Y, wherein n is an integer number selected from0-10; R for each instance is independently hydrogen, alkyl, aralkyl,alkenyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; or twoinstances of R taken together form a 3-6 membered cycloalkyl orheterocycloalkyl containing 1, 2, or 3 heteroatoms selected from N, O,and S; and Y is selected from the group consisting of OR⁴, SR⁴, N(R⁴)₂,—(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, —(NR⁴)S(O)₂OR⁴, and —(CRR²R³), wherein R² ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴,—O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; R³ ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴,—O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; and R⁴for each instance is independently selected from the group consisting ofhydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; or R¹ is a moiety selected from the group consisting of:

and

N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.

While the examples below are generally directed tothiosuccinyl-crosslinked hemoglobin comprising a α₂β₂ tetramerichemoglobin, other forms of hemoglobin are also contemplated by thepresent disclosure, such as other tetrameric hemoglobin, e.g., α₂γ₂;trimeric hemoglobin, e.g., α₃β₂, α₂β, αγ₂, and α₂γ; dimeric hemoglobin,e.g., αβ and αγ; and the like; as well as polymeric forms of hemoglobincomprising one or more monomeric forms of hemoglobin; and hemoglobinderivatives that have been subjected to other methods of chemicalmodification including, but not limited to, methods for conjugation topolyalkylene oxide, reaction with pyridoxal phosphate, reaction with adialdehyde, reaction with bis-diaspirin ester, reaction withiodoacetamide or other thiol-blocking reagents, or reaction in thepresence of reagents such as 2,3-diphosphoglycerate (2,3-DPG) orchemically similar compounds, or genetically crosslinked hemoglobinderivatives, such as 2αβ₂ (dialpha beta hemoglobin), wherein the dialphamoiety comprises two alpha chains that are genetically crosslinked with,e.g., a glycine linker covalently linking the N-terminus and theC-terminus of each alpha chain.

The tetrameric hemoglobin can comprise naturally occurring and/ornon-naturally occurring α, β, and γ globin chain polypeptide sequences.

The tetrameric hemoglobin can be human hemoglobin, bovine hemoglobin,porcine hemoglobin, ovine hemoglobin, equine hemoglobin, or blood fromother invertebrates and recombinant and/or transgenically producedhemoglobin.

In instances in which the tetrameric hemoglobin is human hemoglobin[e.g., comprising two α globin chain (UniProt Accession Number: P69905);and two β globin chains (UniProt Accession Number: P68871)], each N* mayindependently represent a nitrogen present in any one or more of aminoacid residues at position 1, 8, 12, 17, 41, 57, 61, 62, 91, 100, 128,and 140 of the α globin chains; or at position 1, 9, 18, 60, 62, 66, 67,83, 96, 121, 133, and 145 of the β globin chains. In certainembodiments, each N* independently represents a nitrogen present in theamino acid residues at position 100 of the α globin chains.

In instances in which the tetrameric hemoglobin is bovine hemoglobin[e.g., comprising two α globin chain (UniProt Accession Number: P01966);and two β globin chains (UniProt Accession Number: P02070)], each N* mayindependently represent a nitrogen present in any one or more of aminoacid residues at position 1, 8, 12, 17, 41, 57, 62, 69, 91, 100, 128,and 140 of the α globin chains; or at position 1, 7, 16, 18, 58, 60, 64,65, 75, 81, 94, 103, 119, and 131 of the β globin chains. In certainembodiments, each N* independently represents a nitrogen present in anyone or more of amino acid residues and 1 and 81 of the β globin chains.

In instances in which the tetrameric hemoglobin is porcine hemoglobin[e.g., comprising two α globin chain (UniProt Accession Number: P01965);and two β globin chains (UniProt Accession Number: P02067)], each N* mayindependently represent a nitrogen present in any one or more of aminoacid residues at position 1, 7, 11, 16, 40, 56, 61, 68, 90, 99, 127, and139 of the α globin chains; or at position 1, 9, 18, 60, 62, 66, 67, 77,83, 88, 133 and 145 of the β globin chains. The presence of oxygen inthe crosslinking reaction is also known to affect the p50 value of theresulting crosslinked hemoglobin. Depending on the oxygen content in thefumaryl crosslinking reaction, the p50 value of the resultingfumaryl-crosslinked hemoglobin, as well as the thiosuccinyl-crosslinkedhemoglobin, can have a value ranging from 5-70 mmHg.

In certain embodiments, the hemoglobin is crosslinked under oxygenatedconditions, to give a fumaryl-crosslinked hemoglobin, as well as thethiosuccinyl-crosslinked hemoglobin, with a p50 value of 5-20 mmHg or10-20 mmHg. In certain embodiments, the hemoglobin is crosslinked underdeoxygenated conditions, to give a fumaryl-crosslinked hemoglobin, aswell as the thiosuccinyl-crosslinked hemoglobin, with a p50 value of20-70 mmHg; 20-70 mmHg; 30-70 mmHg; 40-70 mmHg; 40-60 mmHg; 38-50 mmHg;45-65 mmHg; or 55-65 mmHg.

In instances in which the hemoglobin is first thioblocked by reaction ofthe hemoglobin with iodoacetamide thereby forming a thioblockedhemoglobin; crosslinking the thus formed thioblocked hemoglobin with thea fumaryl crosslinking agent thereby forming a fumaryl-crosslinkedthioblocked hemoglobin; and contacting the fumaryl-crosslinkedthioblocked hemoglobin with a thiol or a pharmaceutically acceptablesalt or zwitterion thereof thereby forming a thiosuccinyl-crosslinkedthioblocked hemoglobin, the p50 value of the resultingthiosuccinyl-crosslinked thioblocked hemoglobin crosslinked underdeoxygenated conditions can range from 15-50 mmHg; 25-50 mmHg; or 35-50mmHg, while the p50 value of the resulting thiosuccinyl-crosslinkedthioblocked hemoglobin crosslinked under oxygenated condition can rangefrom 5-20 mmHg; 5-15 mmHg, 5-10 mmHg or 10-15 mmHg.

In certain embodiments, R¹ is alkyl or —(CR₂)_(n)Y; or R¹ is a moietyselected from the group consisting of:

and

N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.

In instances in which R¹ is:

m can be 1-1000, 1-900, 1-800, 1-700, 1-600, 1-500, 1-400, 1-300, 1-200,1-100, 1-50, 1-25, 1-20, 1-15, 1-10, or 1-5.

In instances in which R¹ is —(CR₂)_(n)Y, n can be 0-10, 0-9, 0-8, 0-7,0-6, 0-5, 0-4, 0-3, 0-2, 0-1, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,or 1-2. In certain embodiments, each R is independently hydrogen oralkyl. In certain embodiments, R¹ is —(CH₂)_(n)Y.

In certain embodiments, Y is —(CRR²R³), wherein R for each instance isindependently selected from the group consisting of hydrogen, alkyl,aralkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; R² ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —N(R⁴)₂, or —NH(C═O)R⁴; R³ is hydrogen, alkyl, aralkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —CO₂R⁴, —(C═O)NHR⁴,—OR⁴, or —N(R⁴)₂; and R⁴ for each instance is independently selectedfrom the group consisting of hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl. In certain embodiments, R ishydrogen. In certain embodiments, R² is —N(R⁴)₂ or —NH(C═O)R⁴; and R³ is—CO₂R⁴, —(C═O)NHR⁴, —OR⁴, or —N(R⁴)₂.

In certain embodiments, Y is —(CRR²R³), wherein, R is hydrogen; R² ishydrogen, —N(R⁴)₂, —NH(C═O)R⁴, or —NH(C═O)N(R⁴)₂; and R³ is —CO₂R⁴,—(C═O)NHR⁴, —OR⁴, or —N(R⁴)₂.

In certain embodiments, R¹ is —(CH₂)_(n)(CHR²R³), wherein n is 1, 2, 3,or 4; R² is —N(R⁴)₂ or —NH(C═O)R⁴; R³ is —CO₂R⁴ or —(C═O)NHR⁴; and eachR⁴ is independently selected from the group consisting of hydrogen,alkyl, cycloalkyl, aryl, and heteroaryl.

In certain embodiments, R¹ is —(CH₂)_(n)(CHR²R³), wherein n is 1, 2, 3,or 4; R² is —N(R⁴)₂ or —NH(C═O)R⁴; R³ is —CO₂H; and each R⁴ isindependently selected from the group consisting of hydrogen or alkyl.

In certain embodiments, R¹ is selected from the group consisting of:

or a pharmaceutically acceptable salt of zwitterion thereof, wherein mis a whole number selected from 1-1000.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobincomprises a α₂β₂ tetrameric bovine hemoglobin comprising two α globinchains (UniProt Accession Number: P01966) and two β globin chains(UniProt Accession Number: P02070), wherein the β globin chains arecrosslinked with at least one thiosuccinyl crosslinking moiety ofFormula 1, wherein R¹ is selected from the group consisting of:

or a pharmaceutically acceptable salt or zwitterion thereof, wherein atleast one N* represents the nitrogen at the N-terminus of a β globinchain and at least one N* represents the nitrogen in the lysine sidechain at position 81 of a β globin chain.

In alternative embodiments, the present disclosure also provides analogsin which the sulfur depicted in Formula 1 is replaced with selenium,disulfide, and diselenides, wherein R¹ and each N* are as defined in anyone or more embodiments described herein.

In alternative embodiments, the present disclosure also provides athiosuccinyl-crosslinked hemoglobin comprising a tetrameric hemoglobinand at least one thiosuccinyl crosslinking moiety of Formula 1a:

or a conjugate salt or zwitterion thereof, wherein each N* independentlyrepresents a nitrogen selected from the group consisting of a nitrogenin a lysine residue side chain in the hemoglobin and a nitrogen at aN-terminus in the hemoglobin; and R¹ is an alkylthio group.

In certain embodiments, the alkylthio group is a moiety of Formula 2a:

wherein n is an integer selected from the group consisting of 0, 1, 2,3, and 4; R for each instance is independently selected from the groupconsisting of hydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl,aryl, and heteroaryl; R² is hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, —N(R⁴)₂, or —NH(C═O)R⁴; R³ ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —CO₂R⁴, —(C═O)NHR⁴, —OR⁴, or —N(R⁴)₂; and R⁴ for eachinstance is independently selected from the group consisting ofhydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; or the alkylthio group is a moiety selected from the groupconsisting of:

or a conjugate salt thereof, wherein m is a whole number selected from1-1000.

In certain embodiments, n is 1 or 2; R is hydrogen; R² is —NHR⁴, or—NH(C═O)R⁴; and R³ is hydrogen, —OR⁴, —CO₂R⁴, or —(C═O)NHR⁴, wherein R⁴for each instance is independently selected from the group consisting ofhydrogen and alkyl.

In certain embodiments, the alkylthio group is selected from the groupconsisting of:

or a conjugate salt of zwitterion thereof, wherein m is a whole numberselected from 1-1000.

In certain embodiments, the thiosuccinyl-crosslinked hemoglobin isisolated and/or substantially pure. In certain embodiments, thethiosuccinyl-crosslinked hemoglobin is substantially stroma-free.

The present disclosure also provides a pharmaceutical compositioncomprising a thiosuccinyl-crosslinked hemoglobin described herein and atleast one pharmaceutically acceptable excipient and/or pharmaceuticallyacceptable carrier.

The thiosuccinyl-crosslinked hemoglobin described herein and theirpharmaceutically acceptable salts can be administered to a subjecteither alone or in combination with pharmaceutically acceptable carriersor diluents in a pharmaceutical composition according to standardpharmaceutical practice. The thiosuccinyl-crosslinked hemoglobin can beadministered parenterally. Parenteral administration includesintravenous, intramuscular, intraperitoneal, subcutaneous and topical,the preferred method being intravenous administration.

Accordingly, the present disclosure provides pharmaceutically acceptablecompositions, which comprise a therapeutically-effective amount of thethiosuccinyl-crosslinked hemoglobin described herein, formulatedtogether with one or more pharmaceutically acceptable carriers(additives) and/or diluents. The pharmaceutical compositions of thepresent disclosure may be specially formulated for administration inliquid form, including those adapted for the following: (1) parenteraladministration, for example, by intravenous as, for example, a sterilesolution or suspension.

Asset out herein, certain embodiments of the thiosuccinyl-crosslinkedhemoglobin described herein may contain a basic functional group, suchas amino, and are, thus, capable of forming pharmaceutically-acceptablesalts with pharmaceutically-acceptable acids. The term“pharmaceutically-acceptable salts” in this respect, refers to therelatively non-toxic, inorganic and organic acid addition salts ofthiosuccinyl-crosslinked hemoglobin of the present disclosure. Thesesalts can be prepared in situ in the administration vehicle or thedosage form manufacturing process, or by separately reacting a purifiedthiosuccinyl-crosslinked hemoglobin of the invention in its free baseform with a suitable organic or inorganic acid, and isolating the saltthus formed during subsequent purification. Representative salts includethe bromide, chloride, sulfate, bisulfate, carbonate, bicarbonate,nitrate, acetate, valerate, oleate, palmitate, stearate, laurate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate,and laurylsulphonate salts and the like.

The pharmaceutically acceptable salts of the compounds of the presentdisclosure include the conventional nontoxic salts or quaternaryammonium salts of the compounds, e.g., from nontoxic organic orinorganic acids. For example, such conventional nontoxic salts includethose derived from inorganic acids such as hydrochloride, hydrobromic,sulfuric, sulfamic, phosphoric, nitric, and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the thiosuccinyl-crosslinked hemoglobin described hereinmay contain one or more acidic functional groups and, thus, are capableof forming pharmaceutically-acceptable salts withpharmaceutically-acceptable bases. The term “pharmaceutically-acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of the thiosuccinyl-crosslinkedhemoglobin of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives, solubilizing agents, buffers and antioxidants can also bepresent in the compositions.

Methods of preparing the pharmaceutical comprising thethiosuccinyl-crosslinked hemoglobin include the step of bringing intoassociation a thiosuccinyl-crosslinked hemoglobin described herein withthe carrier and, optionally, one or more accessory ingredients. Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association a compound described herein with liquidcarriers (liquid formulation), liquid carriers followed bylyophilization (powder formulation for reconstitution with sterile wateror the like), or finely divided solid carriers, or both, and then, ifnecessary, shaping or packaging the product.

Pharmaceutical compositions of the present disclosure suitable forparenteral administration comprise one or more thiosuccinyl-crosslinkedhemoglobins described herein in combination with one or morepharmaceutically-acceptable sterile isotonic aqueous or non-aqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars (such assucrose), alcohols, non-ionic surfactants (such as Tween 20),antioxidants, buffers, bacteriostats, chelating agents, solutes whichrender the formulation isotonic with the blood of the intended recipientor suspending or thickening agents.

Examples of suitable aqueous and non-aqueous carriers which may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants, such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the compounds of the presentdisclosure may be ensured by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

The pharmaceutical composition may comprise between 3-15 g/dL of thethiosuccinyl-crosslinked hemoglobin. In certain embodiments, thepharmaceutical composition comprises between 4-15 g/dL; 5-15 g/dL; 5-14g/dL; 6-14 g/dL; 7-13 g/dL; 8-12 g/dL; 9-11 g/dL; or 9.5-10.5 g/dL ofthe thiosuccinyl-crosslinked hemoglobin. In certain embodiments, thepharmaceutical composition comprises isolated and substantially purethiosuccinyl-crosslinked hemoglobin.

In certain embodiments, the pharmaceutical composition comprises one ormore thiosuccinyl-crosslinked hemoglobin selected from the groupconsisting of thiosuccinyl-crosslinked hemoglobin comprising one, two,and three thiosuccinyl crosslinking moieties of Formula 1. The number ofdifferent thiosuccinyl-crosslinked hemoglobin present in thepharmaceutical composition and their relative amounts can be readilycontrolled by modifying the reaction conditions of the crosslinkingreaction and/or by separating undesired fumaryl-crosslinked hemoglobincrosslinking and/or thiosuccinyl-crosslinked hemoglobin thiol additionproducts by purification. In certain embodiments, the pharmaceuticalcomposition comprises a thiosuccinyl-crosslinked hemoglobin having onethiosuccinyl crosslinking moiety of Formula 1; athiosuccinyl-crosslinked hemoglobin having two thiosuccinyl crosslinkingmoieties of Formula 1; and a thiosuccinyl-crosslinked hemoglobin havingthree thiosuccinyl crosslinking moieties of Formula 1. In certainembodiments, the pharmaceutical composition comprises athiosuccinyl-crosslinked hemoglobin having one thiosuccinyl crosslinkingmoiety of Formula 1; a thiosuccinyl-crosslinked hemoglobin having twothiosuccinyl crosslinking moieties of Formula 1; and athiosuccinyl-crosslinked hemoglobin having three thiosuccinylcrosslinking moieties of Formula 1 in a mass ratio of 2.8-3.4:5.6-6.2:0.7-1.3; 2.9-3.3:5.7-6.1: 0.8-1.2; 3.0-3.2:5.8-6.0:0.9-1.1; or3.1:5.9:1.0, respectively.

In certain embodiments, the pharmaceutical composition comprises athiosuccinyl-crosslinked hemoglobin having one thiosuccinyl crosslinkingmoiety of Formula 1 at 0.1-99%; 0.1-95%; 0.1-90%; 0.1-80%; 0.1-70%;0.1-60%; 0.1-50%; 10-50%; 20-50%; 20-40%; 25-45%; or 25-35% wt/wt withrespect to the total weight of all of the thiosuccinyl-crosslinkedhemoglobin present in the pharmaceutical composition (e.g., relative tothe total weight of the thiosuccinyl-crosslinked hemoglobin having onethiosuccinyl crosslinking moiety of Formula 1; thethiosuccinyl-crosslinked hemoglobin having two thiosuccinyl crosslinkingmoieties of Formula 1; and the thiosuccinyl-crosslinked hemoglobinhaving three thiosuccinyl crosslinking moieties of Formula 1 present inthe pharmaceutical composition).

In certain embodiments, the pharmaceutical composition comprises athiosuccinyl-crosslinked hemoglobin having two thiosuccinyl crosslinkingmoiety of Formula 1 at 0.1-99%; 0.1-95%; 0.1-90%; 10-90%; 20-90%;20-80%; 20-70%; 30-70%; 40-70%; 50-70%; 50-60%; or 55-65% wt/wt withrespect to the total weight of all of the thiosuccinyl-crosslinkedhemoglobin present in the pharmaceutical composition.

In certain embodiments, the pharmaceutical composition comprises athiosuccinyl-crosslinked hemoglobin having three thiosuccinylcrosslinking moiety of Formula 1 at 0.1-99%; 0.1-95%; 0.1-90%; 0.1-80%;0.1-70%; 0.1-60%; 0.1-50%; 0.1-40%; 0.1-30%; 0.1-20%; 5-20%; or 5-15%wt/wt with respect to the total weight of all of thethiosuccinyl-crosslinked hemoglobin present in the pharmaceuticalcomposition.

The pharmaceutical composition can comprise the fumaryl crosslinkedhemoglobin in less than 10%, less than 9%, less 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, lessthan 1% by weight, less than 0.5%, or less than 0.1% by weight; orsubstantially no fumaryl crosslinked hemoglobin.

The thiosuccinyl-crosslinked hemoglobin and the fumaryl crosslinkedhemoglobin may be present in the pharmaceutical composition in a massratio of 90:10 to 99.99:0.01. In certain embodiments, thethiosuccinyl-crosslinked hemoglobin and the fumaryl crosslinkedhemoglobin may be present in the pharmaceutical composition in a massratio of 91:9 to 99.99:0.01; 92:8 to 99.99:0.01; 93:7 to 99.99:0.01;94:6 to 99.99:0.01; 95:5 to 99.99:0.01; 96:4 to 99.99:0.01; 97:3 to99.99:0.01; 98:2 to 99.99:0.01; 99:1 to 99.99:0.01; 99.5:0.5 to99.99:0.01; or 99.9:0.1 to 99.99:0.01, respectively. In certainembodiments, the pharmaceutical composition comprises substantially nofumaryl crosslinked hemoglobin.

In certain embodiments, the pharmaceutical composition further comprisesan antioxidant. Exemplary antioxidants include, but are not limited to,cysteine, N-acetyl cysteine, γ-glutamyl-cysteine, glutathione,2,3-dimercapto-1-propanol, 1,4-butanedithiol, sodium dithionite, otherbiologically compatible thiols and ascorbate. The antioxidant caninhibit or reverse the formation of methemoglobin.

In certain embodiments, the pharmaceutical composition comprises 5%(w/w) or less of the antioxidant. In certain embodiments, thepharmaceutical composition comprises 4.5% (w/w) or less; 4.0% (w/w) orless; 3.5% (w/w) or less; 3.0% (w/w) or less; 2.5% (w/w) or less; 2.0%(w/w) or less; 1.5% (w/w) or less; 1.0% (w/w) or less; 0.9% (w/w) orless; 0.8% (w/w) or less; 0.7% (w/w) or less; 0.6% (w/w) or less; 0.5%(w/w) or less; 0.4% (w/w) or less; 0.3% (w/w) or less; 0.2% (w/w) orless; or 0.1% (w/w) or less of the antioxidant. In certain embodiments,the pharmaceutical composition comprises between 0.001 to 1% (w/w); 0.01to 1% (w/w); 0.01 to 1% (w/w); 0.01 to 0.9% (w/w); 0.01 to 0.8% (w/w);0.01 to 0.7% (w/w); 0.01 to 0.6% (w/w); 0.01 to 0.5% (w/w); 0.01 to 0.4%(w/w); 0.01 to 0.3% (w/w); 0.05 to 0.3% (w/w); 0.1 to 0.3% (w/w); or0.15 to 0.25% (w/w) antioxidant.

In certain embodiments, the pharmaceutical composition includes lessthan about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% byweight methemoglobin.

In certain embodiments, provided herein is a solid pharmaceuticalcomposition comprising a thiosuccinyl-crosslinked hemoglobin asdescribed herein, NAC, sucrose, and Tween 20.

In certain embodiments, provided herein is a pharmaceutical compositioncomprising a thiosuccinyl-crosslinked hemoglobin as described herein,NAC, NaCl, and sodium acetate. In certain embodiments, thepharmaceutical composition comprising a thiosuccinyl-crosslinkedhemoglobin as described herein, NAC, NaCl, sodium acetate, sucrose, andTween 20.

The present disclosure also provides methods of preparing thethiosuccinyl-crosslinked hemoglobin described herein. Thethiosuccinyl-crosslinked hemoglobin can readily be prepared by anynumber of well-known methods known to those of ordinary skill in theart.

In certain embodiments, the method for preparing thethiosuccinyl-crosslinked hemoglobin comprises: contacting a tetramerichemoglobin with a fumaryl crosslinking agent thereby forming afumaryl-crosslinked hemoglobin; contacting the fumaryl-crosslinkedhemoglobin with a thiol or a pharmaceutically acceptable salt orzwitterion thereof thereby forming the thiosuccinyl-crosslinkedhemoglobin.

Any fumaryl crosslinking agent that is capable of intramolecularlycrosslinking hemoglobin known in the art can be used in the methodsdescribed herein. In certain embodiments, the fumaryl crosslinking agentcan be represented by a compound of Formula 4:

wherein each LG can independently be any leaving group in the art.Exemplary leaving groups include, but are not limited to, Cl, Br, I,3,5-dibromosalicylate, salicylate, or the like.

In certain embodiments, LG is selected from the group consisting of:

The compound of Formula 4 can be performed or formed in situ, e.g., byreaction of fumaric acid with a carbonyl activating agent and optionallya coupling additive.

Exemplary carbonyl activating agents include, but are not limited to,carbodiimide, such as DCC, DIC, EDC, CIC, BMC, CPC, BDDC, PIC, PEC, andBEM, a uronium/aminium salt, such as HATU, HBTU, TATU, TBTU, HAPyU,TAPipU, HAPipU, HBPipU, HAMBU, HBMDU, HAMTU, 5,6-B(HATU), 4,5-B(HATU),HCTU, TCTU, and ACTU, phosphonium salts, such as AOP, BOP, PyAOP, PyBOP,PyOxm, PyNOP, PyFOP, NOP, and PyClock, immonium salts, such as BOMI,BDMP, BMMP, BPMP, and AOMP.

Exemplary coupling additives include, but are not limited to, HOBt.6-NO₂-HOBt, 6-Cl-HOBt, 6-CF₃—HOBt, HOAt, HODhbt, HODhat, HOSu, andOxyma.

In certain embodiments, the crosslinking agent is a salicyl fumarateanalog, wherein the aryl rings of each of the salicyl groups isindependently optionally substituted.

In certain embodiments, the crosslinking agent is selected from thegroup consisting of bis-3,5-dibromosalicyl fumarate (DBSF), fumarylchloride and bis(salicyl) fumarate.

In the step of contacting the crosslinking agent and the tetramerichemoglobin, the molar ratio of the crosslinking agent and the tetramerichemoglobin can be between 0.8:1 to 20:1, respectively. In certainembodiments, the crosslinking agent and the tetrameric hemoglobin arepresent a molar ratio between 0.8:1 to 19:1; 0.8:1 to 18:1; 0.8:1 to17:1; 0.8:1 to 16:1; 0.8:1 to 15:1; 0.8:1 to 14:1; 0.8:1 to 13:1; 0.8:1to 12:1; 0.8:1 to 11:1; 0.8:1 to 10:1; 0.8:1 to 9:1; 0.8:1 to 8:1; 0.8:1to 7:1; 0.8:1 to 6:1; 0.8:1 to 5:1; 0.8:1 to 4:1; 0.8:1 to 3.5:1; 0.8:1to 3:1; 0.8:1 to 2.5:1; 0.8:1 to 2:1; 0.8:1 to 1.5:1; 1:1 to 3:1; 1.1:1to 3:1; 1.5:1 to 3:1; 2:1 to 3:1; or 2.25:1 to 2.75:1, respectively.

In the step of contacting the crosslinking agent and the tetramerichemoglobin, the concentration of the tetrameric hemoglobin can bebetween 5-25 g/dL. In certain embodiments, the concentration of thetetrameric hemoglobin in the step of contacting the crosslinking agentand the tetrameric hemoglobin can be between 5-20 g/dL; 10-20 g/dL;10-18 g/dL; 10-16 g/dL; 10-15 g/dL; 11-15 g/dL; 12-15 g/dL; or 13-15g/dL.

The tetrameric hemoglobin can be reacted with the crosslinking agent ina polar protic solvent, such as in an aqueous solution. In certainembodiments, the crosslinking reaction takes place in water.

In order to facilitate the crosslinking reaction, the pH of the reactionsolvent can be maintained at a pH greater than 7. In certainembodiments, the pH of the crosslinking reaction solvent has a pHbetween 7-10; 8-10; 8.5 to 9.5; 8.7 to 9.3; or 8.9 to 9.1.

The thus formed fumaryl-crosslinked hemoglobin can optionally purifiedusing any method known to those skilled in the art, such as byfiltration, heat-induced precipitation, centrifugation, chromatography,and the like.

The fumaryl-crosslinked hemoglobin can then reacted with the thiolthereby forming the thiosuccinyl-crosslinked hemoglobin.

The thiol can be represented by the formula R¹SH as defined in anyembodiment described herein.

The fumaryl-crosslinked hemoglobin can be present in the reaction withthe thiol at a concentration between 5-20 g/dL. In certain embodiments,the fumaryl-crosslinked hemoglobin is present in the reaction with thethiol at a concentration between 5-18 g/dL; 5-16 g/dL; 5-14 g/dL; 5-12g/dL; 7-12 g/dL; 8-12 g/dL; or 9-11 g/dL.

The thiol can be present in the reaction with the fumaryl-crosslinkedhemoglobin at a concentration between 1-500 mM. In certain embodiments,the thiol can be present in the reaction with the fumaryl-crosslinkedhemoglobin at a concentration between 1-450 mM; 1-400 mM; 1-350 mM;1-300 mM; 1-250 mM; 1-200 mM; 1-180 mM; 1-160 mM; 1-140 mM; 1-120 mM;1-100 mM; 10-100 mM; 20-100 mM; 30-100 mM; 30-90 mM; 40-80 mM; 77.5-310mM, 174-3110 mM, 9.7-77.5 mM; 19.4-77.5 mM; or 38.8-77.5 mM.

The reaction of the thiol and the fumaryl-crosslinked hemoglobin can beconducted at a pH between 7-11. In certain embodiments, the reaction ofthe thiol and the fumaryl-crosslinked hemoglobin is conducted at a pHbetween 7-11; 7-10; 7.4 to 10; 7.4 to 9, 7.4 to 8.2, or 8.2 to 9. The pHof the thiol addition reaction solvent can be maintained at the desiredpH by use of pH buffer within the desired range or the addition of aBrønsted base to the reaction mixture, as needed. The selection of theappropriate Brønsted base or pH buffer is well within the skill of aperson of ordinary skill in the art. Useful Brønsted bases include, butare not limited to Group I and Group II hydroxides, carbonates, andbicarbonates; organic amines, and the like.

The fumaryl-crosslinked hemoglobin can be reacted with the thiol in apolar protic solvent, such as in an aqueous solution. In certainembodiments, the thiol addition reaction takes place in water.

The reaction of the thiol with the fumaryl-crosslinked hemoglobin cangenerally conducted until all of the fumaryl-crosslinked hemoglobinstarting material is converted to the desired thiosuccinyl-crosslinkedhemoglobin, the fumaryl-crosslinked hemoglobin no longer is beingconverted to the desired thiosuccinyl-crosslinked hemoglobin, and/or theconcentration of impurities and/or side products increases beyond adesired amount. Depending on the reaction conditions, the reaction ofthe thiol with the fumaryl-crosslinked hemoglobin can take between 1-72hr; 6-72 hr, 12-72 hr, 24-72 hr, 36-72 hr, 48-72 hr, 60-72 hr, 12-48 hr,or 24-48 hr. In cases in which the rate of reaction of the thiol withthe fumaryl-crosslinked hemoglobin is very slow (e.g., such as in thecase of certain high molecular weight PEGylated thiols), the reaction ofthe thiol with the fumaryl-crosslinked hemoglobin can take up to onemonth.

The thus formed thiosuccinyl-crosslinked hemoglobin can optionallypurified using any method known to those skilled in the art, such as byfiltration, heat-induced precipitation, centrifugation, chromatography,and the like.

The present disclosure also provides therapeutic methods of using thethiosuccinyl-crosslinked hemoglobin described herein. Thethiosuccinyl-crosslinked hemoglobin can be used in any therapeuticmethods that hemoglobin based oxygen carriers can be used.

The present disclosure provides a method for increasing the volume ofthe blood circulatory system in a subject in need thereof, wherein themethod comprises transfusing into the system of the subject atherapeutically effective amount of the thiosuccinyl-crosslinkedhemoglobin according to any embodiment or combination of embodimentsdescribed herein. In certain embodiments, the subject suffers fromhemorrhagic shock.

The present disclosure provides a method of supplying oxygen to thetissues and organs in a subject in need thereof, wherein the methodcomprises transfusing into the system of the subject a therapeuticallyeffective amount of the thiosuccinyl-crosslinked hemoglobin according toany embodiment or combination of embodiments described herein. Incertain embodiments, the subject suffers from ischemia, including forexample myocardial ischemia-reperfusion injury. The ischemia can beglobal or regional.

The present disclosure provides a method of treating cancer in a subjectin need thereof, wherein the method comprises transfusing into thesystem of the subject a therapeutically effective amount of thethiosuccinyl-crosslinked hemoglobin according to any embodiment orcombination of embodiments described herein. Thethiosuccinyl-crosslinked hemoglobin can be administered alone or incombination with one or more cancer therapeutics and/or radiotherapy totreat cancer.

In certain embodiments, the cancer is selected from the group consistingof leukemia, head and neck cancer, colorectal cancer, lung cancer,breast cancer, liver cancer, nasopharyngeal cancer, esophageal cancerand brain cancer. In certain embodiments, the cancer is triple-negativebreast cancer or colorectal cancer.

The cancer therapeutic can be bortezomib, 5-fluorouracil, doxorubicin,or cisplatin.

The present disclosure also provides a method of treating systemic lupuserythematosus in a subject in need thereof, wherein the method comprisestransfusing into the system of the subject a therapeutically effectiveamount of the thiosuccinyl-crosslinked hemoglobin according to anyembodiment or combination of embodiments described herein.

The present disclosure also provides a method of treating peripheralartery disease in a subject in need thereof, wherein the methodcomprises transfusing into the system of the subject a therapeuticallyeffective amount of the thiosuccinyl-crosslinked hemoglobin according toany embodiment or combination of embodiments described herein.

The present disclosure also provides a method of treating traumaticbrain injury in a subject in need thereof, wherein the method comprisestransfusing into the system of the subject a therapeutically effectiveamount of the thiosuccinyl-crosslinked hemoglobin according to anyembodiment or combination of embodiments described herein.

EXAMPLES Example 1: Process Overview

An exemplary schematic flow diagram of the process of makingcysteinyl-succinyl crosslinked hemoglobin, is illustrated in FIG. 2.Bovine whole blood collected from the slaughter house was processed,lysed and purified by ultrafiltration step and column chromatography toproduce highly purified hemoglobin solution (PHS). To prevent thedissociation of the hemoglobin into heterodimer, the tetramerichemoglobin was stabilized by crosslinking reaction with DBSF while theresidual DBSF and hydrolyzed derivatives were removed by ultrafiltrationstep to bring the DBSF and 3-5, dibromosalicylic acid (DBSA) levels tobelow 0.03% (w/w). The hemoglobin crosslinked by fumaryl bridges(fumaryl-crosslinked hemoglobin) was modified by 1,4-addition reactionof cysteine with the fumaryl moieties present in the fumaryl-crosslinkedhemoglobin to form cysteinyl-succinyl crosslinked hemoglobin with <2%methemoglobin level under deoxygenated conditions and followed by anultrafiltration purification step to bring the cysteine and cystinelevels to below 0.03% (w/w). To reduce the level of met-hemoglobin,N-acetyl cysteine at a concentration of 0.05% to 0.2% was added to thesolution containing the above-mentioned cysteinyl-succinyl crosslinkedhemoglobin.

Example 2: Preparation of Highly Purified Bovine Hemoglobin Solution

Blood cells were separated from the whole bovine blood throughcentrifugation and the collected blood cells were subjected to a cellwashing step (Lima, M. C., 2007, Artif Cells Blood Substit ImmobilBiotechnol, 35(4):431-47). In certain embodiments, the method for theisolation and purification of hemoglobin from blood cells described inthe literature can be used to prepare the hemoglobin used in the currentmethod (Houtchens, R. A. & Rausch, C. W., 2000, U.S. Pat. No. 6,150,507;Wong, B. L. & Kwok, S. Y., 2011, U.S. Pat. No. 7,989,593 B1). Theresidual amount of plasma was further removed from the collected bloodcells by hollow fiber filtration step. A hypotonic solution was mixedwith the washed blood cells to release the intracellular hemoglobinthrough a tightly controlled process. The cell debris were removed fromcell lysate via a 0.2 μm filtration step and followed by additionalultrafiltration steps to partially remove the impurities to form apartially purified hemoglobin solution. To further purify the hemoglobinsolution, the hemoglobin solution was buffer exchanged to containminimal salt concentration prior to the negative mode anion columnchromatography step. The flow through fraction containing highlypurified hemoglobin was collected for which the pH, tHb and saltconcentration were adjusted, sterile filtered and stored at 2-8° C.prior to the downstream process. With a tight process control for thepreparation of stroma-free hemoglobin, the quality and purity of thehighly purified hemoglobin were analyzed and are summarized in Table 1.

TABLE 1 Level of Contaminants and Impurities in Highly PurifiedHemoglobin Solution. Contaminants/Impurities Mycoplasma UndetectablePhospholipids Phosphatidyl- BLOD (LOD = ethanolamine 0.52 μg/mL)Phosphatidyl- BLOD (LOD = serine 1.73 μg/mL) Sphingomyelin BLOD (LOD =0.42 μg/mL) Total Phospholipid <9.2 nmol/mL Endotoxin <0.1 EU/mLResidual Bovine DNA <0.025 pg/μl Protein Impurities Bovine 0.17 ppmImmunoglobulin G Bovine <0.02 ppm Serum Albumin Bovine 0.02 ppm PlasmaProteins Bovine 36.04 ppm Carbonic Anhydrase LOD: Limit of Detection;BLOD: Below Limit of Detection

Example 3: Preparation of Fumaryl-Crosslinked Tetrameric Hemoglobin

The crosslinking reaction was carried out under deoxygenated conditions,that is, less than 0.1 mg/L dissolved oxygen level in 0.9% w/v aqueousNaCl solution. DBSF was added to the highly purified bovine hemoglobinsolution to form fumaryl-crosslinked hemoglobin. This stabilizationprocedure stabilizes the tetrameric form of hemoglobin (˜65 kDa), whichprevents the dissociation into dimeric forms, which are excreted throughthe kidneys. The stabilization of the tetrameric hemoglobin was carriedout through a reaction of highly purified hemoglobin (tHb=13-15 g/dL)with 2.5 molar equivalents of DBSF at pH 9.0 for a period of 4 hours at10-30° C. under an inert atmosphere of nitrogen (dissolved oxygen levelmaintained at less than 0.1 mg/L) to prevent oxidation of the hemoglobinto form ferric methemoglobin, which is physiologically inactive. Duringthe reaction, the reaction pH was maintained by the addition ofdeoxygenated 0.1-0.5 M NaOH solution. The reaction mixture was thenpurified through tangential flow filtration using 30 kDa nominalmolecular weight cut off (NMWCO) membrane. In the purification process,the concentration of the hemoglobin solution was maintained at 9.5-10.5g/dL through a continuous feeding of acetate buffer (99 mM NaCl, 46 mMsodium acetate) into the reaction tank. The purification was completedafter undergoing 10-16 diafiltration volume. The purity of thefumaryl-crosslinked tetrameric hemoglobin obtained was determined bysize exclusion chromatography (SEC).

Example 4: Determination of Hemoglobin Stabilization Using SizeExclusion Chromatography

A UPLC system (ACQUITY UPLC H-class System) equipped with a PDA detectorand a size exclusion column (ACQUITY UPLC Protein BEH SEC column, 200 Å,1.7 um, 4.6 mm×300 mm) was equilibrated with Tris-MgCl₂ Buffer (50 mMTris, 750 mM MgCl₂ and 0.116 mM EDTA-Na₂, pH 6.5) at flow rate of 0.25mL/min for 400 minutes. Sample (3 mg/mL) was freshly prepared with waterand analyzed by SEC with the detection wavelength set as 280 nm. Underthis column condition, all the non-crosslinked bovine hemoglobindissociated into dimeric forms, as depicted in FIG. 3A, while thecrosslinked hemoglobin showed as tetramer with minor amounts of octamer,as depicted in FIG. 3B. From the SEC chromatogram, the elution peaksobserved in the retention time of 9.4, 10.8 and 11.5 min are the proteinsignals of the octameric, tetrameric and dimeric form of hemoglobin,respectively. The percentage of different forms of hemoglobin wasquantified by the integrated intensity of the corresponding peaks. Uponcompletion of the crosslinking reaction, the reaction mixture cancontain at least 85% by weight of crosslinked tetrameric bovinehemoglobin (65 kDa), less than 10% by weight crosslinked octamerichemoglobin, and less than 5% by weight dimeric hemoglobin, as shown inFIG. 3B. The tetrameric structure of the bovine fumaryl-crosslinkedhemoglobin molecule contained at least 1 to 3 crosslinker(s) between thebeta globin chains (β-β crosslink), as shown in FIG. 4A.

Example 5 Modification of β-β Crosslink by Thiol-Containing Reagents

Selective modification of the fumaryl moieties of the crosslinker(s) inthe fumaryl-crosslinked hemoglobin was achieved using thiol-containingmolecules, such as cysteine, homocysteine, NAC and 2-mercaptoethaol(BME). Advantageously, the cysteine residues at position 92 of betaglobin chain did not react with the fumaryl moieties.

Example 5A: Modification of β-β Crosslink by Cysteine

In this embodiment, the fumaryl moieties of the crosslinker between thebeta chains were modified by cysteine. In the modification step, 40-80mM cysteine at pH 8.0-8.3 was incubated with fumaryl-crosslinkedhemoglobin (tHb=7-10 g/dL) in acetate buffer (99 mM NaCl, 46 mM sodiumacetate, pH 8.2-8.4) for a period of 15-30 hours at 10-30° C. underdeoxygenated condition for which the dissolved oxygen (DO) levelsmaintained below 0.1 mg/L. The residual cysteine/cystine in the reactionmixture was removed by a filtration step using a 30 kDa NMWCO membranefor which the reaction mixture went through 10-16 diafiltration volume(DV) with acetate buffer to bring the cysteine/cystine levels below0.03% (w/w), as shown in Table 2. Apart from the cysteine/cystinelevels, the levels of the DBSF and its hydrolyzed derivative (DBSA) inthe cysteinyl-succinyl crosslinked hemoglobin were also below 0.03%(w/w).

TABLE 2 Level of Cysteine and Cystine in Cysteinyl-succinyl CrosslinkedBovine Hemoglobin after Filtration Step. Batch Number Cysteine % (w/w)Cystine % (w/w) C8002 <0.0289 (BLOQ) <0.0289 (BLOQ) C8003 <0.0289 (BLOQ)<0.0289 (BLOQ) C8005 <0.0289 (BLOQ) <0.0289 (BLOQ) BLOQ: Below Limits ofQuantification

The completion of the cysteine modification on the fumaryl-crosslinkedhemoglobin was evaluated by ESI-MS analysis. A UPLC system (Agilent6460) equipped with an electrospray ionization triple quadrupole massspectrometer and a C3 column (Agilent, Poroshell 300SB-C3, 5 μm, 1.0mm×75 mm) was equilibrated with acetonitrile with 0.1% formic acid at aflow rate of 0.2 mL/min for 30 minutes. Samples (0.3 mg/mL) were freshlyprepared with water and analyzed by ESI-MS system using positive ionmode. The ESI-MS mass spectra of the samples were obtained by thedeconvolution of the corresponding TIC chromatogram. A 1:1stoichiometric addition of the cysteine to the β-β crosslinked globinchains was found at saturation, as depicted in FIG. 4B. As shown in FIG.4A, the stabilization of hemoglobin via crosslinking between the βglobin chains by DBSF resulted in 3 major species containing 1, 2, and 3crosslink bridges and having the molecular weight of around 31988 Da,32068 Da and 32148 Da, respectively. A cysteine amino acid (121 Da) wascovalently bonded to the fumaryl moieties of these β-β crosslinkedglobin chains, resulting in species of 32,109 Da, 32,310 Da and 32,511Da, respectively, as shown in FIG. 4B.

The sample was further analyzed by ESI-MS/MS analysis, in order toconfirm the fumaryl moiety of the β-β crosslink of thefumaryl-crosslinked hemoglobin was modified by the cysteine amino acid.Sample was analyzed by 10% SDS-PAGE and visualized using 0.1% CoomassieBrilliant Blue R-250, 20% (v/v) methanol and 10% (v/v) acetic acid. Theprotein band corresponding to the major crosslinked globin chain with−32 kDa was excised from the SDS-PAGE gel, cut into cubes (1×1 mm), anddestained with 50% acetonitrile/20% 50 mM ammonium bicarbonate solution.The destained gel cubes were in-gel digested with 10 ng/μL sequencinggrade trypsin in 50 mM ammonium bicarbonate at 37° C. overnight. Aftertrypsin digestion, the trypsin-digested peptides were extracted bydiffusion into 50% (v/v) acetonitrile and 1% (v/v) trifluoroacetic acid.The supernatant was collected and the solvent was removed by SpeedVac at45° C. The trypsin-digested peptides were dissolved in 0.1% (v/v) formicacid, separated by reverse phase C18 column and analyzed usingOrbitrap-Velos Mass Spectrometer. The EIS-MS/MS data was input into thepLink search engine for the crosslink sites analysis. As shown in FIG.5, a peptide crosslinked at the N-terminal nitrogen at position 1 andthe lysine side chain nitrogen at position 81 of β globin chains wasidentified. Importantly, the molecular weight of the crosslink peptidewas 201 Da instead of 80 Da (fumaryl moiety), confirming that thecysteine amino acid (MW=121 Da) was covalently conjugated into thefumaryl moiety of the β-β crosslink peptide, but not the thiol group ofcysteine at position 92 of beta globin.

A novel hemoglobin analog comprising cysteinyl-succinylatedcrosslinker(s) was produced by the above process. The pharmaceuticalcomposition containing cysteinyl-succinyl crosslinked hemoglobinproduced was kept under nitrogen with the presence of 0.2% (w/w) NACwith the following product characteristics: tHb=9.5-10.5 g/dL, pH7.4-8.4, O₂Hb ≤10%, MetHb ≤5%, endotoxin ≤0.25 EU/mL andcysteinyl-succinyl crosslinked hemoglobin in range of 90-100%.

Example 5B: Comparison of Modification of β-β Crosslink byThiol-Containing Reagents

When reacting the fumaryl moieties with other thiol-containing reagentssuch as cysteine, homocysteine and 2-mercaptoethanol, at pH 8.2,different extents of modification between the fumaryl moieties and thethiol reagents were observed. Preliminary studies of thiol-containingreagents were selected based on the steric and electronic effects(pKa_(SH) value), which includes, but is not limited to cysteine,β-mercaptoethanol and homocysteine.

Degassed thiol reagents including cysteine (pKa_(SH)=8.35),β-mercaptoethanol (pKa_(SH)=8.87), and homocysteine (pKa_(SH)=9.6) atdifferent concentrations (111 μL; 1000, 775, 258 and 86 mM in RA⁻buffer, pH=8.2) was added to 1 mL of deoxygenated fumaryl-crosslinkedhemoglobin solution (9.0 g/dL, pH=8.2) (Pitman, I. H. & Morris, I. J.,1979, Aust J Chem, 32: 1567-73). At 3, 6 and 24 hour, samples werecollected and excess thiol was removed using desalting column (150 μL toBio-Spin P6; 2 times). The samples were analyzed using ESI-MS, asdepicted in FIG. 6 and the results are summarized in Table 3.

TABLE 3 Reactivity of Fumaryl-crosslinked Hemoglobin with DifferentThiols. Thiol-containing Reagent Cysteine β-mercaptoethanol HomocysteineConcentration (mM) (mM) (mM) (mM) 8.6 25.8 77.5 8.6 25.8 77.5 100 8.625.8 77.5 Reaction 3 hr x x x x x x x x x x Time 6 hr x x x x x x x x xx 24 hr x x ✓ x x x ✓ x x x x: Incomplete saturation of fumaryl-thiolcoupling reaction ✓: Saturation of fumaryl-thiol coupling reaction

The extent of fumaryl-thiol reaction between the fumaryl-crosslinkedhemoglobin and the thiols progressed in a time and concentrationdependent manner. Unmodified fumaryl-crosslinked hemoglobin remaineddetectable after 3 and 6 hour incubation with thiols at differentconcentrations. At 24 hour, the reaction with 77.5 mM cysteine and 100mM β-mercaptoethanol completely modified all the fumaryl moieties of theβ-β crosslinks in the fumaryl crosslinked-hemoglobin, while unmodifiedfumaryl-crosslinked β-β globin chains remained detectable in thehemoglobin mixture for those incubated with lower concentration ofcysteine, β-mercaptoethanol and homocysteine.

These results indicated that the fumaryl moieties of the β-β crosslinksunderwent highly efficient Michael addition (1,4-addition) reactionswith thiols. Reaction conditions such as pH, salt concentration,equivalents of the thiols and duration of the reaction were optimizedresulting in achieving a 90-100% conversion of the fumaryl-crosslinkedhemoglobin to the desired thiosuccinyl-crosslinked hemoglobin.

It is noteworthy that such modification between the crosslinker of thecrosslinked hemoglobin and thiols, such as cysteine or NAC, did notoccur with hemoglobin that was crosslinked with bis-3,5-dibromosalicylsuccinate (DBSS), as depicted in FIG. 7, as expected.

Example 6: Reaction Studies of Fumaramide with Alkylthiols Under AqueousCondition

The confirmation of the reaction between fumaramide with alkylthiolunder aqueous condition was carried out using the model moleculeN,N′-bis(1-((tert-butoxycarbonyl)amino)-1-(carboxy)pentyl)fumaramide(BocLysF; FIG. 8a ). Upon reaction of BocLysF (15 mM) with modelalkylthiols (77.5 mM) including β-mercaptoethanol and cysteine underaqueous conditions (0.1 M phosphate buffer, pH=8.2, deoxygenated) for 7days, the MS spectra of reaction mixture clearly showed the completeconsumption of starting material BocLysF and the appearance of expectedmass peaks of thiol-addition products (M+H*; 649 and 692 Da,respectively). As shown from the NMR analysis of the reaction mixture,the loss of fumaramide double bond signal, formation of succinamide withgeminal proton coupling peaks on C2 carbon and the splitting of aminesignal were found. These observations can be rationalized by theoccurrence of an addition reaction between fumaramide and alkylthiolthrough thiol-Michael-addition pathway that a pair of geminal protonswas introduced to the C2 carbon of the resulting succinamide.Importantly, the reaction mixture was clean and the expected succinamideand residual alkylthiols were observed in NMR spectra, suggesting thereaction of the fumaryl and alkylthiol was complete. Thus, it is anideal reaction for the post-crosslink modification and functionalizationof fumaryl-crosslinked proteins including hemoglobin. Fullcharacterization data of fumaramide BocLysF and the resultingsuccinamide includingN,N′-bis(1-((tert-butoxycarbonyl)amino)-1-(carboxy)pentyl)-1-(2-hydroxyethylthio)-succinamide(BocLysF-BME; FIG. 8b ) andN,N′-bis(1-((tert-butoxycarbonyl)amino)-1-(carboxy)pentyl)-1-(S-cysteinyl)succinamide(BocLysF-Cys; FIG. 8c ) is shown in Table 4-6.

TABLE 4 The Complete Assignment of ¹H and ¹³C Signals of BocLysF ind6-DMSO. ¹H [ppm] ¹³C [ppm] CH(COOH)(NHCOOC(CH ₃)₃) 1.36 28.70CH(COOH)(NHCOOC(CH₃)₃) 3.74 54.47 CH(COOH)(NHCOOC(CH₃)₃) 6.66 — CH₂CH₂CH₂CH₂NH 1.58 31.30 CH₂ CH ₂CH₂CH₂NH 1.27 23.40 CH₂CH₂ CH ₂CH₂NH1.39 29.13 CH₂CH₂CH₂ CH ₂NH 3.10 38.91 CH₂CH₂CH₂CH₂NH 8.39 — CH═CH 6.77133.47 

TABLE 5 The Complete Assignment of ¹H and ¹³C Signals of BocLysF-BME ind6-DMSO. ¹H [ppm] ¹³C [ppm] CH CH ₂CH₂CH₂CH₂NH 1.55 32.86 CHCH₂ CH₂CH₂CH₂NH 1.21 22.62 CHCH₂CH₂ CH ₂CH₂NH 1.34 20.96 CHCH₂CH₂CH₂ CH ₂NH2.96 39.35 CHCH₂CH₂CH₂CH₂N H 7.88, 8.04 — CH(NHCOOC( CH ₃)₃)(COOH)] 1.3628.69 CH (NHCOOC(CH₃)₃)(COOH)] 3.69 59.87 CH(N H COOC(CH₃)₃)(COOH)] 6.17—

2.31, 2.56 38.99

3.60 43.57

2.61 34.56

3.48 61.15

TABLE 6 The Complete Assignment of ¹H and ¹³C Signals of BocLysF-Cys ind6-DMSO. ¹H [ppm] ¹³C [ppm] CH CH ₂CH₂CH₂CH₂NH 1.55 33.41 CHCH₂ CH₂CH₂CH₂NH 1.20 22.26 CHCH₂CH₂ CH ₂CH₂NH 1.35 29.31 CHCH₂CH₂CH₂ CH ₂NH2.90, 3.13 39.98 CHCH₂CH₂CH₂CH₂N H 8.16, 8.43 — CH (NHCOOC(CH₃)₃)(COOH)]3.63 56.22 CH(NHCOOC( CH ₃)₃)(COOH)] 1.34 28.55 CH(N HCOOC(CH₃)₃)(COOH)] 6.05 —

2.38, 2.52 37.90

3.71 42.87

2.75, 2.94, 3.06 34.26

3.46 55.37

Example 7: Purity of Cysteinyl-Succinyl Crosslinked Hemoglobin inPharmaceutical Composition

As described in Example 5, pharmaceutical composition containingcysteinyl-succinyl crosslinked hemoglobin was produced by the reactionof cysteine with the fumaryl moieties of the β-β crosslinks of thefumaryl-crosslinked hemoglobin.

The purity of the cysteinyl-succinylcrosslinked hemoglobin produced bythe above embodiments was evaluated by ESI-MS analysis. Samples wereanalyzed by LC-MS on an Agilent 6540 Electrospray IonizationQuadrupole-Time-of-Flight spectrometer connected to an liquidchromatography system (Agilent 6460) with a C3 column (Agilent Poroshell300SB-C3, 5 μm, 1.0 mm×75 mm) and the mass spectra were deconvolutedusing the Maximum Entropy algorithm in the Agilent MassHunterQualitative Analysis software. Crosslinked species were identified bymatching the molecular masses from the deconvoluted MS data totheoretical figures. Relative abundances of molecular species wereestimated using the area-under-curve of the deconvoluted spectra.

Based on the analysis of ESI-MS spectrum, the fumaryl-crosslinkedhemoglobin were found to contain one (1XL), two (2XL) or three (3XL)fumaryl crosslinks present in a molar ratio of 28%, 58% and 15%,respectively, in which at least 1 fumaryl bridge was crosslinked betweenβ globin chains. Following the modification with cysteine stated, thepeaks corresponding to the mass of unmodified fumaryl-crosslinked β-βglobin chains were undetectable in the ESI-MS spectrum. Instead, it wasfound that the peaks were found to be present in similar proportion, butthe molecular weights were shifted by 121, 242, and 363 Da, whichcorresponded to the stoichiometric addition of 1 (1XL+1Cys), 2(2XL+2Cys) and 3 cysteine amino acids (3XL+3Cys), respectively. Themolecular weights for the β-β crosslinks before and after cysteinemodification were shown for each component. The findings supported thenotion that the fumaryl thiol coupling reaction between the cysteine andfumaryl-crosslinked hemoglobin was totally complete and at least 95% orhigher conversion was achieved after modification with cysteine underthe tested reaction conditions, as depicted in Table 7.

TABLE 7 Proportion of Beta-Beta Globin Chain (β-β) inFumaryl-crosslinked Hemoglobin Before and After Cysteine Modification. %Hemoglobin with Different Numbers of % Hemoglobin withCysteinyl-succinyl Molecular Different Numbers of Crosslinks AfterCrosslink Species Mass Fumaryl Crosslinks Modification by CysteineFumaryl Crosslink in 1 Fumaryl Crosslink (XL) 31988 Da 28% NA β-β globinChain 2 Fumaryl Crosslinks (2XL) 32068 Da 58% NA 3 Fumaryl Crosslinks(3XL) 32148 Da 15% NA Cysteinyl-succinyl 1 Cysteinyl-succinyl 32109 Da 0% 31% Crosslink in β-β Crosslinker (1XL + 1Cys) Globin Chain 2Cysteinyl-succinyl 32310 Da  0% 59% Crosslinkers (2XL + 2Cys) 3Cysteinyl-succinyl 32511 Da  0% 10% Crosslinkers (3XL + 3Cys) NA: NotApplicable

Example 8: Effects of PH and Reaction Medium on the Reaction Rate of theFumaryl-Thiol Coupling Reaction

The reaction rates of the fumaryl-thiol coupling reaction under otherreaction parameters including pH and reaction medium were examined. Adegassed solution of β-mercaptoethanol (111 μL; 775 mM in RA⁻ or RA⁻with extra 0.9% NaCl) was added to 1 mL of deoxygenatedfumaryl-crosslinked hemoglobin solution (8.7 μg/dL, pH=7.4, 8.2, or 9.0;In RA⁻ or RA⁻ with extra 0.9% NaCl). At 6 and 24 hour, samples werecollected and excess thiol was removed using a desalting column (150 μLto Bio-Spin P6; 2 times). The samples were analyzed using ESI-MSanalysis and the results are shown in Table 8. Complete saturation ofthe fumaryl bridges was not detected in the samples collected at 6hours. Among the reaction conditions at different pH values and saltconcentrations, modification of the fumaryl bridges by ˜77.5 mMβ-mercaptoethanol was complete in 24 hours at pH 7.4 or pH 9.0. Althougha complete modification was achieved with 77.5 mM cysteine at pH 8.2,the modification by 77.5 mM β-mercaptoethanol at pH 8.2 remainedincomplete and the presence of the unmodified fumaryl bridges wasdetected even after 24 hours of incubation. The result also revealedthat there was no observable effect on the reaction rate of the couplingreactions when the salinity of the reaction medium was increased.

TABLE 8 Effects of pH and Reaction Medium on the Reaction Rate of theFumaryl-thiol Coupling Reaction Between Fumaryl- crosslinked Hemoglobinand β-mercaptoethanol. Reaction pH Reaction Condition Reaction Time 7.48.2 9.0 77.5 mM β-mercaptoethanol 6 hr x x x in RA−* Buffer 24 hr ✓ x ✓□77.5 mM β-mercaptoethanol 6 hr x x x in RA−* Buffer with extra 0.9% NaCl24 hr ✓ x ✓ x: Incomplete saturation of fumaryl-thiol coupling reaction✓: Saturation of fumaryl-thiol coupling reaction *Ringer's Acetate MinusBuffer (RA− Buffer)

Example 9: Effects of Thiol-Containing Reagent Concentration andReaction Time on the Reaction Rate of the Fumaryl-Thiol CouplingReaction

A degassed solution of β-mercaptoethanol (111 μL; 775, 388, 194, 97, and48 mM in RA⁻, pH 9.0) or cysteine (111 μL; 775, 388, 194, and 97 mM inRA⁻, pH=9.0) was added to 1.0 mL of deoxygenated fumaryl-crosslinkedhemoglobin solution (9.0 g/dL, pH=9.0). At 6, 24, 48, and 72 hour,samples were collected and excess thiol was removed using desaltingcolumn (150 μL to Bio-Spin P6; 2 times). All the samples were analyzedusing ESI-MS and the results are summarized in Table 9. Thefumaryl-thiol reaction rate increased in a concentration dependentmanner while the coupling rate of cysteine and β-mercaptoethanol to thefumaryl moieties were doubled by increasing the concentration from 19.4mM to 38.8 mM. Complete modification of the fumaryl moieties by eithercysteine or β-mercaptoethanol was achieved within 24 hour. Although thereaction rate was substantially decreased in low thiol reagentsconcentration such as 9.7 mM, saturation of fumaryl bridges was achievedby extending the reaction time to 72 hour.

TABLE 9 Effects of Thiol Concentration and Reaction Time on the ReactionRate of the Fumaryl-thiol Coupling Reaction Between Fumaryl- crosslinkedHemoglobin and Thiols (Cysteine and β-mercaptoethanol). Cysteineβ-mercaptoethanol Concentration (mM) 9.7 19.4 38.8 77.5 4.8 9.7 19.438.8 77.5 Reaction 6 hr x x x x x x x x x Time 24 hr x x ✓ ✓ x x x ✓ ✓48 hr x ✓ ✓ ✓ x x ✓ ✓ ✓ 72 hr ✓ ✓ ✓ ✓ x ✓ ✓ ✓ ✓ x: Complete saturationof fumaryl-thiol coupling reaction ✓: Saturation of fumaryl-thiolcoupling reaction

Complete modification of the fumaryl moieties may be limited by thephysical-chemical properties of the thiol reagents and suboptimalcoupling conditions. Since the reaction conditions leading to thesaturation of the fumaryl moieties varies between different thiolreagents, the inherent molecular properties, such as the steric andelectronic effects, and the reaction conditions, at least act incombination with the equivalents of the thiol reagents, reaction pH andduration, not only affect the rate of modification but also the overallconversion of the coupling reaction. For instance, 77.5, 174 and 310 mMN-acetyl cysteine (NAC) were incubated with fumaryl-crosslinkedhemoglobin at pH 8.2, the samples collected at 24 and 48 hour wereanalyzed by ESI-MS. The results showed that the conversion offumaryl-thiol coupling reaction was found to below 95% for allconcentrations at 24 hour. The fumaryl-thiol coupling reaction at 95%was observed only when extending the incubation time to 48 hour using310 mM NAC. When compared to the fumaryl-thiol reaction using cysteine,the concentration of NAC to saturate the fumaryl bridges were 4 timeshigher and the incubation time was doubled. These results suggest thathigher NAC concentration and incubation time may be required toachieve >95% conversion of the fumaryl moieties of the crosslinkedhemoglobin by NAC. These results suggest that unless a person havingordinary skill in the art was intentionally attempting to carry out thefumaryl-thiol coupling reaction and endeavored to optimize the reactionconditions to ensure complete reaction, the typical concentration ofthiol reagents utilized as an excipient to reduce or inhibit theformation of methemoglobin would not result in the formation ofthiosuccinyl-crosslinked hemoglobin at high levels of conversion (Table10).

TABLE 10 Effects of NAC Concentration and Reaction Time on the ReactionRate of the Fumaryl-thiol Coupling Reaction. NAC Concentration ReactionTime 77.5 mM 174 mM 310 mM 24 hours x X X 48 hours x x ✓□ x: Incompletesaturation of fumaryl-thiol coupling reaction ✓: Saturation offumaryl-thiol coupling reaction

Example 10: Change of p50 Value of Fumaryl-Crosslinked Hemoglobin afterModification of Thiol-Containing Reagents

The oxygen affinity properties of hemoglobin can be described by its p50value, determined from the oxygen dissociation curve. The oxygendissociation curve of the hemoglobin shows the relationship between thehemoglobin saturation at different oxygen tensions, and the p50 is theoxygen tension at which hemoglobin is 50% saturated.

The p50 value of the thiosuccinyl-crosslinked hemoglobin produced by thecomplete reaction between fumaryl-crosslinked hemoglobin and differentthiol-containing reagents was evaluated. The oxygen dissociation curvefor the hemoglobin solution was obtained using a Hemox analyzer (TCSScientific, New Hope, Pa.). Oxygen tension was measured with a Clarkoxygen electrode, and the hemoglobin saturation was measured using abuilt-in dual wavelength spectrophotometer. The measurement was carriedout in Hemox solution (135 mM NaCl, 5 mM KCl and 30 mM TES, pH 7.4) witha final hemoglobin concentration of 0.05 g/dL and the temperaturemaintains at 37° C. throughout the measurement. A computer-basedanalysis of oxygen dissociation curve was performed yielding p50 foroxygen binding. Oxygen dissociation parameters were derived by fittingthe Adair equations to each oxygen dissociation curve by nonlinearleast-squares procedure included in the Hemox analyzer software (TCSHemox DAQ System, Version 2.0).

The results reveal that over 95% of fumaryl-crosslinked hemoglobin wasconverted to the thiosuccinyl-crosslinked hemoglobin by incubatingfumaryl-crosslinked hemoglobin at 310 mM NAC for 48 hour and its p50value increased by 11%. In contrast, a thiosuccinyl-crosslinkedhemoglobin solution containing over 95% purity prepared by incubatingwith either 77.5 mM cysteine or 100 mM β-mercaptoethanol showedcomparable p50 values, compared to the unmodified fumaryl-crosslinkedhemoglobin, as shown in Table 11.

TABLE 11 p50 Values of Thiosuccinyl-crosslinked Hemoglobin AfterModification with Different Thiol Reagents. Fumaryl-Thiosuccinyl-crosslinked Hemoglobin crosslinked β- Incubation HemoglobinN-acetyl Cysteine mercaptoethanol Cysteine Time — 77.5 mM 174 mM 310 mM100 mM 77.5 mM 0 hr 55 mmHg — — — — — 24 hr — 56 mmHg 60 mmHg 63 mmHg 53mmHg 54 mmHg 48 hr — 58 mmHg 62 mmHg 61 mmHg — —

The results also reveal that a complete modification was achieved byconjugating the cysteine to thiol-blocked fumaryl-crosslinked hemoglobin(hemoglobin in which the position 92 cysteine residue of the betaglobins is blocked (alkylated) by reaction with iodoacetamide prior tothe crosslinking reaction with the fumaryl crosslinking agent) accordingto the preparation procedure as described in Example 5A. The p50 valueof the cysteinyl-succinyl crosslinked thiol-blocked hemoglobin alsoremained unchanged after modification either for that having a p50 valueof ˜36 mmHg (crosslinked under deoxygenated condition) or ˜9 mmHg(crosslinked under oxygenated condition), as shown in Table 12. Thisreveals that the conjugation of cysteine to the fumaryl-crosslinkedhemoglobin having different oxygen carrying capacities surprisingly didnot alter their p50 values.

TABLE 12 p50 Values of Cysteinyl-succinyl Crosslinked HemoglobinProduced from Thiol-blocked Fumaryl-crosslinked Hemoglobin EitherCrosslinked Under Deoxygenated or Oxygenated conditions. Thiol-blockedThiol-blocked Cysteinyl-succinyl Fumaryl-crosslinked Crosslinked BovineHemoglobin Bovine Hemoglobin Crosslink Reaction 37 mmHg 35 mmHg underDeoxygenated Condition Crosslink Reaction 9 mmHg 9 mmHg under OxygenatedCondition

Example 11: In-Vitro Stability of Cysteinyl-Succinyl CrosslinkedHemoglobin

The stability of the cysteinyl-succinylated moieties in thecysteinyl-succinyl crosslinked hemoglobin was tested in the presence ofsmall molecule thiols such as cysteine, NAC, or glutathione (GSH). Thesesmall molecule thiols are commonly used in hemoglobin-based therapeuticdrug formulations as excipients, e.g., to reduce the methemoglobinlevels.

The fumaryl-crosslinked hemoglobin and cysteinyl-succinyl crosslinkedhemoglobin were incubated with NAC, cysteine, or GSH at 1:4 or 1:8 molarratios (100 mg/mL hemoglobin) under an apoxic environment. Samples takenat various time points were analyzed by ESI-MS on an Agilent 6540Electrospray Ionization Quadrupole-Time-of-Flight spectrometer connectedto a liquid chromatography system (Agilent 6460) with a C3 column(Agilent Poroshell 300SB-C3, 5 μm, 1.0 mm×75 mm). The mass spectra weredeconvoluted using the Maximum Entropy algorithm in the AgilentMassHunter software. Crosslinked globin species were identified bymatching the molecular masses from the deconvoluted MS data totheoretical figures. Relative abundances of molecular species wereestimated using the area-under-curve of the deconvoluted spectra.

Table 13 shows the estimation of relative abundances of differentcrosslinked β-β species % derived from ESI-MS data of cysteinyl-succinylcrosslinked hemoglobin over 12 months in the presence of NAC at aneight-fold molar excess. The area under curve (AUC) of the maincrosslinked species remained stable after at least 12 months, showingthe stability of the abundances of the crosslinked species over time.

TABLE 13 Stability of β-β Globin Chains in Cysteinyl-succinylCrosslinked Hemoglobin in the Presence of NAC over 12 Months. %Hemoglobin with Different Numbers of Cysteinyl-succinyl β-βCrosslinksTime 1XL + 1Cys 2XL + 2Cys 3XL + 3Cys At Release 32% 58% 10%  1 Month39% 51% 11%  2 Months 36% 53% 11%  5 Months 40% 53% 6% 7 Months 42% 51%7% 10 Months 39% 55% 7% 11 Months 35% 57% 8% 12 Months 37% 56% 7%

Percentages are relative abundances of the different crosslinkedspecies, as estimated from deconvoluted LC-MS spectra. “XL”—crosslink,“Cys”—cysteine.

In contrast, the fumaryl moieties of the fumaryl-crosslinked hemoglobinreacted continuously with NAC in a 9-week monitoring period, with theNAC covalently attached to the β-β crosslink of the hemoglobin molecule(Table 14). Similar reactions with cysteine and GSH were also evident(Table 15 &16).

TABLE 14 Stability of β-β Globin Chains in Fumaryl-crosslinkedHemoglobin in the Presence of 0.2% NAC over 9 Weeks. % Hemoglobin withDifferent Species of β-β Crosslinks 1XL + 2XL + 2XL + Time 1XL 2XL 1NAC1NAC 2NAC Day 3 18% 20%  20% 37% 5% 1 Week 19% 21%  20% 37% 3% 3 Weeks12% 9% 28% 43% 8% 5 Weeks  9% 3% 32% 44% 11%  9 Weeks  3% 0% 38% 41%19% 

Percentages are relative abundances of the different crosslinkedspecies, as estimated from deconvoluted LC-MS spectra. XL”—crosslink,“NAC”—N-acetyl cysteine.

TABLE 15 Stability of β-β Globin Chains in Fumaryl-crosslinkedHemoglobin with 4-fold Molar Excess of Cysteine over 4 Hours. %Hemoglobin with Different Species of β-β Crosslinks Time 1XL + 1Cys 2XL2XL + 1Cys 2XL + 2Cys T = 0.25 Hour 20% 58% 22%  0% T = 4 Hours 22% 24%34% 20%

TABLE 16 Stability of β-β Globin Chains in Fumaryl-crosslinkedHemoglobin with 4-fold Molar Excess of Glutathione over 4 Hours. %Hemoglobin with Different Species of β-β Crosslinks Time 1XL 1XL + 1GSH2XL 2XL + 1GSH T = 0.25 hour 27% 4% 64%  5% T = 4 hours 20% 9% 58% 12%

Percentages are relative abundances of the different crosslinkedspecies, as estimated from deconvoluted LC-MS spectra. “XL”—crosslink,“GSH”—glutathione.

Example 12: Stability of NAC as Excipient in Cysteinyl-SuccinylCrosslinked Hemoglobin Solution

Thiol-containing compounds are commonly used in hemoglobin-based oxygentherapeutics as excipients for conversion and prevention ofdysfunctional methemoglobin. In this experiment, NAC was used as anexcipient in cysteinyl-succinyl crosslinked hemoglobin solution, and NACwas oxidized to N,N′-diacetyl-L-cystine (NAC₂) when reduced thedysfunctional methemoglobin to functional hemoglobin form. Samples weretaken at various time points over storage and the level of NAC and itsoxidation product, NAC₂, in the cysteinyl-succinyl crosslinkedhemoglobin solution were measured by reverse phase liquidchromatography. For the measurement, samples were treated with 5%meta-phosphoric acid to precipitate the proteins, and the supernatantwas separated on a XBridged C18 column (5 m, 4.6 mm×250 mm), using anisocratic elution in buffer containing 100 mM sodium phosphate pH 2.3,5.7 mM sodium 1-octanesulfonate:methanol 91:9 (v/v). NAC and NAC₂ werequantified using their respective calibration curves ranging from 9.375μg/mL to 300 μg/mL.

In contrast to the total level of NAC and NAC₂ in fumaryl-crosslinkedhemoglobin solution (FIG. 9A), cysteinyl-succinyl crosslinked hemoglobinhad stable levels of thiol excipient NAC over an extended period oftime, as depicted in FIG. 9B.

Example 13: Functional Stability of Cysteinyl-Succinyl CrosslinkedHemoglobin

The p50 values of the fumaryl-crosslinked hemoglobin andcysteinyl-succinyl crosslinked hemoglobin solution in the presence of0.2% NAC (v/v) were measured using the Hemox analyzer (TCS ScientificCorp). Hemoglobin product samples at 0.5 mg/mL in Hemox buffer (pH 7.4)were oxygenated by bubbling oxygen through for 30 min and thendeoxygenated by bubbling nitrogen through until the p02 reached 1.9mmHg. The resulting oxygen equilibrium curves were analyzed using theTCS software, using the Adair equation to adjust for incompleteoxygenation of the sample being measured.

Cysteinyl-succinyl crosslinked hemoglobin had a stable p50 value over atleast 3 months post-production. In contrast, fumaryl-crosslinkedhemoglobin showed a 12% increase in p50 values over 10 weeks presumablydue to covalent binding of NAC to the β-β crosslinks of the crosslinkedhemoglobin molecule, as depicted in Table 17.

TABLE 17 p50 Stability of Fumaryl-crosslinked Hemoglobin andCysteinyl-succinyl Crosslinked Hemoglobin, in the Presence of 0.2% NAC,respectively. Adair's p50 (mmHg) Fumaryl-crosslinked Cysteinyl-succinylCrosslinked Time Hemoglobin Hemoglobin At Release 64 67 1 Month 66 67 3Month 70 67

Example 14: In-Vivo Stability of Cysteinyl-Succinyl CrosslinkedHemoglobin

To assess the in-vivo stability of hemoglobin products, male SpragueDawley rats were anesthetized by isoflurane (5% for induction, 1-2% formaintenance) and either fumaryl-crosslinked hemoglobin orcysteinyl-succinyl crosslinked hemoglobin was infused intravenously viathe femoral vein at 0.8-2.0 mL per hour, at a dose level of 620 mg/kg.0.5-1 mL blood samples were collected from the femoral artery 2 hourspost infusion into heparin tubes. Hemoglobin products were enriched fromplasma samples by strong anion exchange chromatography. Plasma sampleswere diluted 80000 times in 20 mM Tris-HCl pH 8.9 and loaded onto aHiTrap Q HP column (GE Healthcare) equilibrated in 20 mM Tris-HCl pH8.9. Hemoglobin products were eluted using a HiTrap Q HP column (GEHealthcare) over a gradient of 0-400 mM NaCl in 20 mM Tris-HCl pH 8.9.Enrichment of hemoglobin products were assessed by sodium dodecylsulfate polyacrylamide gel electrophoresis. Pooled fractions wereanalyzed by ESI-MS using an Agilent 6540 Electrospray IonizationQuadrupole-Time-of-Flight spectrometer.

As depicted in FIG. 10A, the β-β crosslinks of cysteinyl-succinylcrosslinked hemoglobin did not undergo further modifications with thiolsin-vivo compared to pre-infusion. In contrast, those offumaryl-crosslinked hemoglobin product were modified with cysteine andNAC in-vivo (FIG. 10B). Cysteine is an important redox regulator inplasma, and NAC is an excipient in the hemoglobin product formulations.These results show the in-vivo stability of the cysteinyl-succinylcrosslinked hemoglobin.

Example 15: Restoration of Tissue Oxygenation in Hemorrhagic Shock(Fumaryl-Crosslinked Hemoglobin Vs. Cysteinyl-Succinyl CrosslinkedHemoglobin)

Liver tissue oxygenation was evaluated in a severe hemorrhagic shockmodel in Sprague Dawley rat as follows:

Group 1: Fumaryl-crosslinked Hemoglobin Solution (650 mg Hb/kg of bodyweight); and

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (650 mgHb/kg of body weight).

Sprague Dawley rats were anesthetized and instrumented for surgicalprocedure. Laparotomy was performed by a large middle incision to exposethe liver. A large area oxygen sensor (LAS, Oxford Optronix, UK) wasinserted between the right lobe and triangle lobe of the rat liver andallowed for stabilization. After collection of baseline oxygen tensionlevel, rats were rendered hypotensive by hemorrhage in order to triggeroxygen supply/demand imbalance as reflected by an elevated arteriallactate level (8 to 11 mM/L) and an arterial base excess <−12 mM/L.Following the induction of shock and meeting the entry criteria, ratswere administered with infusion of either 650 mg/kg fumaryl-crosslinkedhemoglobin solution (Group 1) or cysteinyl-succinyl crosslinkedhemoglobin solution (Group 2). The liver oxygen tension levels of these2 hemoglobin molecules were monitored and compared up to 1 hourpost-infusion.

As shown in FIG. 11, results indicate that the infusion of 650 mg/kgcysteinyl-succinyl crosslinked hemoglobin solution (Group 2) showed asignificant increase in liver tissue oxygenation, comparing tofumaryl-crosslinked hemoglobin solution (Group 1) throughout theexperiment.

This reveals that the cysteinyl-succinyl crosslinked hemoglobin has asuperior oxygen off-loading capability in ischemic/hypoxic conditions,compared to conventional fumaryl-crosslinked hemoglobin.

Example 16: Restoration of Blood Perfusion in Ischemic Limb(Fumaryl-Crosslinked Hemoglobin Vs. Cysteinyl-Succinyl CrosslinkedHemoglobin)

Restoration of blood flow in ischemic limb was evaluated in mice byperforming femoral artery ligation mimicking peripheral artery disease.

Group 1: Negative Control (Volume-matched RA− buffer, n=8)

Group 2: Fumaryl-crosslinked Hemoglobin Solution (1600 mg/kg, n=2)

Group 3: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (1600 mg/kg,n=3)

To induce critical limb ischemia, ICR (CD-1) mice were anesthetized toperform surgical procedure. The femoral artery was ligated to the distalpoint where it bifurcated into the saphenous and popliteal arteries. Atpost 24 hours arterial ligation, mice were administrated with differenttreatments via bolus tail vein injection. Serial Laser Doppler Imaginganalysis (Moor instruments, Devon, UK) was performed to monitor bloodflow at baseline, right after ligation, Day 7 post treatment, Day 14post treatment and Day 21 post treatment and the mean blood flow fromthe knee to toe was quantified and calculated. Tivi600 Tissue viabilityimager modulating with Tivi106 Oxygen Mapper analyzer (WheelsBridge AB,Sweden) was performed to monitor change in oxygenated hemoglobin atbaseline, right after ligation, 30 minutes post treatment, 60 minutespost treatment, Day 7 post treatment, Day 14 post treatment and Day 21post treatment. The mean change in oxygenated Hb (Oxy-Hb) from the kneeto toe was quantified and calculated.

As shown in FIG. 12, there was significant increase in Oxy-Hb in groupof mice receiving cysteinyl-succinyl crosslinked hemoglobin treatmentwas compared with RA-buffer group (negative control) at 30 minutespost-treatment (p<0.05), whereas fumaryl-crosslinked hemoglobintreatment did not show significant increase in Oxy-Hb as compared withRA− buffer group. At Day 21 post-treatment, mice treated with bothcysteinyl-succinyl crosslinked hemoglobin solution (110.5±7.8%, p<0.01vs control) and fumaryl-crosslinked hemoglobin solution (106.0±2.6%,p<0.05 vs control) showed significantly higher level of Oxy-Hb than RA−buffer (74.6±12.7%).

As shown in FIG. 13, there was a significant improvement of ischemiclimb blood flow in the group of mice receiving cysteinyl-succinylcrosslinked hemoglobin solution (34.9±3.0%, p<0.001) andfumaryl-crosslinked hemoglobin solution (29.3±2.0%, p<0.05) as comparedwith RA− buffer group (negative control) at Day 7 post-treatment, asimilar trend of significant increase in perfusion with thecysteinyl-succinyl crosslinked hemoglobin treatment was observed fromDay 7 onward. At Day 21 post-treatment, mice treated withcysteinyl-succinyl crosslinked hemoglobin solution (56.5±9.6%, p<0.001vs control) resulted in a more significant improvement in bloodperfusion compared with stabilized fumaryl-crosslinked hemoglobinsolution (41.6±4.3%, p<0.05 vs control), and RA− buffer (23.5±9.7%).

To gain mechanistic insights into the “sustained” improvement of Oxy-Hband perfusion on Day 21, circulating mesenchymal stem cell populationswere analyzed by flow cytometry at different time points post treatment.As shown in FIG. 14, results indicated an increase in CD45⁻CD29⁺,CD45⁻CD105⁺, CD45⁻CD106⁺ MSC populations were observed followinginduction of limb ischemia and which sustained for a longer period oftime up to Day 21 comparing with control. Consistent with therestoration in perfusion, a more significant increase in MSC populationswas observed following cysteinyl-succinyl crosslinked hemoglobintreatment.

Collectively, cysteinyl-succinyl crosslinked hemoglobin showed bettertreatment effect than conventional fumaryl-crosslinked hemoglobin, interms of oxygenated hemoglobin, blood flow and circulating mesenchymalstem cell populations in peripheral artery disease model.

Example 17: Method of Using Stabilized Cysteinyl-Succinyl CrosslinkedHemoglobin

The cysteinyl-succinyl crosslinked hemoglobin solution of the presentdisclosure was used for improving the delivery of oxygen and treatmentagainst global and regional ischemic/hypoxic conditions includinghemorrhagic shock, myocardial ischemia reperfusion injury, peripheralartery disease and traumatic brain injury. In addition, thecysteinyl-succinyl crosslinked hemoglobin solution was also used fortreating autoimmune diseases and cancer treatment as follows:

Hemorrhagic Shock: a restoration of tissue oxygenation and mean arterialpressure in hemorrhagic shock;

Peripheral Artery Disease: a significant restoration of blood flow andincrease in Oxy-Hb level in critical limb ischemia;

Myocardial Ischemia Reperfusion Injury: a significant reduction ofmyocardial infraction in heart;

Systemic Lupus Erythematosus: a significant reduction in immune complexformation in tissues/organs and amelioration of tissue/organ damages;

Traumatic Brain Injury: a significant improvement in both neurologicaland motor functions and a reduction of TBI-induced astrocyte activationin controlled cortical impact (CCI) induced traumatic brain injury; and

Cancer Treatment: a significant inhibition of tumor growth intriple-negative breast cancer (TNBC) and colorectal cancer xenograftmodel, respectively.

The dosage of cysteinyl-succinyl crosslinked hemoglobin is approximately100-1600 mg/kg.

Example 18: Treatment of Severe Hemorrhagic Shock in Cynomolgus Monkey

Cysteinyl-succinyl crosslinked hemoglobin was used for the treatment ofsevere hemorrhagic shock in cynomolgus monkey.

Muscle tissue oxygenation and mean arterial pressure were evaluated in asevere hemorrhagic shock model in cynomolgus monkey as follows:

Group 1: Autologous Plasma (Positive control, equivalent volume ofcysteinyl-succinyl crosslinked hemoglobin administrated) (n=2)

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (500 mg/kgof body weight) (n=2)

Cynomolgus monkeys were anesthetized and instrumented for surgicalprocedure. A needle encased oxygen sensor (Oxford Optronix, UK) wasinserted in triceps and allowed to stabilize. A blood pressure sensor(Biopac Systems Inc, US) was inserted in the left femoral artery andallowed to stabilize. After collection of baseline oxygen tension leveland mean arterial pressure, monkeys were rendered hypotensive byhemorrhage to decrease mean arterial pressure to 20 mmHg and keptmaintaining the mean arterial pressure ranging from 20-24 mmHg for 60minutes. Following the induction of shock and meeting the criteria,monkeys were administered with infusion of either autologous plasma(positive control) or cysteinyl-succinyl crosslinked hemoglobinsolution. The muscle oxygenation tension level and mean arterialpressure of these two groups were measured and compared up to 3 hourpost-infusion.

As shown in FIG. 15, results indicate the infusion of 500 mg/kgcysteinyl-succinyl crosslinked hemoglobin solution resulted in anincrease in muscle tissue oxygenation after resuscitation and keptbetter restoration at post 3 hours of resuscitation. For mean arterialpressure restoration, infusion with the cysteinyl-succinyl crosslinkedhemoglobin solution resulted in a better restoration of mean arterialpressure than autologous plasma treatment at post 3 hours time point ofresuscitation, as shown in FIG. 16.

This reveals that cysteinyl-succinyl crosslinked hemoglobin increasedtissue oxygenation and maintained a better restoration of mean arterialpressure in cynomolgus monkey with severe hemorrhagic shock.

Example 19: Treatment of Peripheral Artery Disease in Mice

Cysteinyl-succinyl crosslinked hemoglobin was used for blood perfusionand the delivery of oxygen in attenuated critical limb ischemia in mice.

Restoration of blood flow in ischemic limb was evaluated in mice byperforming femoral artery ligation mimicking peripheral artery disease.In this study, 32 mice were randomly assigned into 4 groups, 8 mice ineach group.

Group 1: RA− Buffer (Negative Control, volume-matched to Group 4);

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (400 mgHb/kg of body weight);

Group 3: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (800 mgHb/kg of body weight); and

Group 4: Cysteinyl-succinyl Crosslinked Hemoglobin (1600 mg Hb/kg ofbody weight).

To induce critical limb ischemia, ICR (CD-1) mice were anesthetized toperform surgical procedure. The femoral artery was ligated to the distalpoint where it bifurcates into the saphenous and popliteal arteries. Atpost 24 hours arterial ligation, mice were administrated with differenttreatments via bolus tail vein injection. Serial laser Doppler imaginganalysis (Moor instruments, Devon, UK) was performed to monitor bloodflow at baseline, right after ligation, Day 7 post treatment, Day 14post treatment and Day 21 post treatment and the mean blood flow fromthe knee to toe was quantified and calculated. Tivi600 Tissue viabilityimager modulating with Tivi106 Oxygen Mapper analyzer (WheelsBridge AB,Sweden) was performed to monitor change in oxygenated hemoglobin atbaseline, right after ligation, 30 minutes post treatment, 60 minutespost treatment, Day 7 post treatment, Day 14 post treatment and Day 21post treatment. The mean change in oxygenated Hb from the knee to toewas quantified and calculated.

As shown in FIG. 17, there was a significant and dose-dependent increasein Oxy-Hb in all group of mice receiving cysteinyl-succinyl crosslinkedhemoglobin (Hb) as compared with RA− buffer group (Group 1) at 30minutes post-treatment (800 mg/kg Hb, *p<0.05; 1600 mg/kg Hb, *p<0.05).At Day 21 post-treatment, mice treated with 400 mg/kg Hb (102.3±4.0%, p***<0.001), 800 mg/kg Hb (125.7±32.3%, **p<0.01) and 1600 mg/kg Hb(128.0±30.5%, **p<0.01) showed significantly higher level of oxygenatedHb than RA− buffer (74.6±12.7%).

As shown in FIG. 18, there was a significant improvement of ischemiclimb blood flow in all group of mice receiving cysteinyl-succinylcrosslinked hemoglobin solution (Group 2-4) as compared with RA− buffergroup (Group 1) from Day 7 post-treatment onward (400 mg/kg Hb,**p<0.01; 800 mg/kg Hb, p<**0.01; 1600 mg/kg Hb, ***p<0.001). At Day 21post-treatment, mice treated with 400 mg/kg Hb (36.5±8.5%, *p<0.05), 800mg/kg Hb (45.7±14.7%, **p<0.01) and 1600 mg/kg Hb (61.0±15.2%,***p<0.001) showed higher significant blood flow than RA− buffer(23.5±9.7%).

To gain mechanistic insights into the “sustained” improvement of Oxy-Hband perfusion on Day 21, circulating mesenchymal stem cell populationswere analyzed by flow cytometry at different time points post treatment.As shown in FIG. 19, results indicate an increase in CD45⁻CD29⁺,CD45⁻CD105⁺, CD45⁻CD106⁺ MSC populations were observed followinginduction of limb ischemia and which sustained for a longer period oftime up to Day 21 comparing with negative control group. This wasconsistent with the observation of the significant increase in Oxy-Hband perfusion at Day 21 post-treatment.

Collectively, the experimental data demonstrates that cysteinyl-succinylcrosslinked hemoglobin activated circulating mesenchymal stem cells andresulted in a functional restoration of perfusion and the delivery ofoxygen in attenuated critical limb ischemia in mice.

Example 20: Treatment of Myocardial Ischemia-Reperfusion Injury in Rats

Cysteinyl-succinyl crosslinked hemoglobin was used as a cardiacprotective agent to reduce the myocardial infarct size in a rat model ofacute myocardial ischemia-reperfusion. Twelve rats were randomly dividedinto 2 groups as follows:

Group 1: Lactated Ringer's Solution (Control Group); and

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (100 mgHb/kg of body weight).

Myocardial ischemia and reperfusion were established in a standard ratmodel. Briefly, anaesthesia was induced in Wistar rats using isoflurane.Animals were then placed on a heated rodent operating table and internaltemperature was continuously monitored with a rectal probe. Animalsunderwent bladder catheterization to monitor urine output, right jugularcannulation to allow fluid infusion, left carotid cannulation for bothcontinuous arterial pressure monitoring and blood withdrawal, andtracheostomy. After insertion of a peritoneal catheter through akey-hole laparotomy, anaesthesia was maintained with repeated bolus ofthiopental sodium 5 mg/kg and analgesia was assured with buprenorphine0.1 mg/kg sc. A fluid bolus of 10 ml/kg was provided before induction ofmechanical ventilation (tidal volume 12 ml/kg, PEEP 3 cmH₂O, RR 80 bpm)and a continuous infusion of a 50-50 mix of Lactated Ringer'ssolution+glucose 2.5% (10 ml/kg/h) was used as maintenance.

At the end of the surgical phase, after a recovery period of 60 minpost-instrumentation, a left-sided thoracotomy was performed between thefourth and fifth ribs. A snare was placed around the region ofmyocardium containing the left anterior descending (LAD) coronary arteryand ischaemia induced by tightening it. The ischaemic phase lasted 40min of acute ligature followed by 120 min of reperfusion. 100 mg/kg ofthe cysteinyl-succinyl crosslinked hemoglobin solution or LactatedRinger's solution was infused at 20 mins post-ischaemia and continueduntil 20 mins post-reperfusion at a rate of 1.5 ml/kg/h. Heart rate,mean arterial blood pressure, and temperature were measured continuouslyand degree of myocardial infarction/injury was assessed histologicallyas infarction size as a proportion of area at risk.

As shown in FIG. 20, infusion of 100 mg/kg cysteinyl-succinylcrosslinked hemoglobin solution demonstrated a significant reduction inmyocardial infarct size when compare to control group. Importantly, inthis study when animals were administered intravenously withcysteinyl-succinyl crosslinked hemoglobin solution, there was no adverseeffect on blood pressure and hemodynamic throughout the experiment,either during the ischemia and reperfusion period.

Example 21: Renal Protective Effect in Systemic Lupus ErythematosusMouse Model

Cysteinyl-succinyl crosslinked hemoglobin was used for the treatment ofSystemic Lupus Erythematosus (SLE) in mice.

A chromatin-immunized lupus mouse model was used to examine thetherapeutic effect of cysteinyl-succinyl crosslinked hemoglobin solutionon lupus nephritis. Activated lymphocyte-derived DNA (ALD-DNA) wasprepared from sorting-purified apoptotic immune cells. As shown in FIG.21, murine lupus nephritis was induced in C57BL/6 mice immunized withALD-DNA (100 g/mouse) dissolved in Freund's compete adjuvant on Day 1.Boost emulsion of ALD-DNA (50 g/mouse) with Freund's incomplete adjuvantwas performed on Day 14 and 28. Cysteinyl-succinyl crosslinkedhemoglobin was administrated intravenously at 800 mg/kg on Day 1, 4, 8,and 11 in a 2 week treatment period.

After 12-week disease progression, the mice were scarified and thekidneys were harvested, fixed and sectioned. Renal damage assessment wasperformed by staining the kidney tissue slides with hematoxylin andeosin (H&E) while glomerular immune complex deposition assessment wasperformed by immunofluorescence staining of immune complex deposition(IgM and IgG) using fluorochome-labelled antibodies. The scoring systemis shown as follows:

0=normal/no signal

1=mild cellular disruption/weak signal in less than 25% of glomeruli

2=moderate cellular disruption and appearance of balloon cells andvacuolation/moderate signal in greater than 50% of glomeruli

3=extensive cell disruption and vacuolation/extensive strong signal ingreater than 50% of glomeruli. Glomerular activity index is assessedwith the parameters below in a totality of score 0-15.

a) Cellular proliferation (0-3)

b) Leukocyte infiltration (0-3)

c) Fibrinoid necrosis or karyorrhexis (0-3)

d) Cellular crescents (0-3)

e) Hyaline thrombi, wire loops (0-3)

Significant reduction in glomerular IgG and IgM deposition was observedin the treatment with cysteinyl-succinyl crosslinked hemoglobin solutionwhen compared to control (FIG. 22). Moreover, a significantly lower thancontrol in total glomerular activity index was observed, showing lesscellular proliferation, fibrinoid necrosis and cellular crescent in thekidneys treated with cysteinyl-succinyl crosslinked hemoglobin solution(FIG. 23).

In conclusion, cysteinyl-succinyl crosslinked hemoglobin significantlyameliorates the development of lupus nephritis in mice.

Example 22: Treatment of Severe Traumatic Brain Injury in Rat

Cysteinyl-succinyl crosslinked hemoglobin was used for the treatment oftraumatic brain injury (TBI) produced by controlled cortical impact inrat model. The effect of treatment with cysteinyl-succinyl crosslinkedhemoglobin solution administrated as a single dose on functional andhistopathological outcomes following TBI was evaluated. In this study,21 Sprague Dawley Rats were randomly assigned into 2 groups as follows:

Group 1: Saline Buffer (Negative Control); and

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (620 mgHb/kg of body weight).

Each rat was implanted with a femoral vein catheter with BUTONVAB95BS(Instech) button about 14 days before the start of the experiment andhoused in pairs in Opti-Rat cages at a 12:12 hour light/dark cycle withfood and water offered adlibitum for at least 7 days. To induce severetraumatic brain injury by open-skull controlled cortical impact (CCI),rats received either 1.2 mg/kg sustained release buprenorphine(Buprenorphine SR™ Lab) plus 0.03 mg/kg regular buprenorphine(Vetergesic) or 1.2 mg/kg sustained release buprenorphine subcutaneously30 min or 24 hours before, were anesthetized to perform surgicalprocedure. The skull was exposed by a single incision along the midlineusing a sterile scalpel-blade then, a 6 mm circular craniotomy wasperformed using a micro drill device (Harvard Apparatus, 72-6065) in theleft parietal bone at the center of the coronal, sagittal and lambdoidsutures used as the outermost boundaries to expose the intact dura.Trephination site was then filled with sterile saline until ready forthe impact. Exposed cortex with intact dura was subjected to controlledcortical impact injury. A controlled cortical impact was produced on theexposed cortex by the impactor actuator mounted on the Leica Impact One™device with a 5.0 mm diameter impactor rod-tip, impacting the cortex atthe velocity of 4.0 m/s, and the depth of the cortical deformation setat 3.00 mm with a dwell time of 200 ms to produce injury within the leftparietal cortex.

At the end of CCI procedure, the wound was cleaned using sterile salinethen, the skin wound was closed using single interrupted sutures and therat was allowed to recover. No supplemental oxygen was given after TBI.One hour after CCI, the rats received a single infusion of eithercysteinyl-succinyl crosslinked hemoglobin solution at 620 mg/kg (Group2, n=10) or equivalent volume of saline (Group 1, n=11).

Neurological functions were assessed using a 21-point neuroscore at 4hours and at Day 1, 3, and 7 following TBI and sensory-motor functionswere also assessed by cylinder and horizontal ladder tests at Day 3 and7, respectively. Brains were harvested on Day 7 post-TBI and processedfor histochemical analysis.

Single severe TBI induced neurological and motor deficits in 1-3 daysfollowed by spontaneous recovery by Day 7 post-TBI in both treatmentgroup (Group 2) and saline control group (Group 1). Interestingly,treatment with cysteinyl-succinyl crosslinked hemoglobin solutionimproved both neurological and motor functions to a significant degree,compared to the saline control group. The result shows that a singledose of 620 mg/kg cysteinyl-succinyl crosslinked hemoglobin solution,one hour after CCI resulted in a better neurological score at Day 1 (26%improvement in the neuroscore, *p<0.05), as depicted in FIG. 24.Concurrently, treatment with cysteinyl-succinyl crosslinked hemoglobinsolution, one hour after CCI also significantly improved performance ofTBI animals measured by the horizontal ladder test, as compared to thesaline control group on Day 3 (18% reduction in ladder error score,*p<0.05). In contrast, the saline control group reached the samerecovery level but only at Day 7 (*p<0.05, as compared to Day in thesame group), indicating that the effect of the cysteinyl-succinylcrosslinked hemoglobin on accelerating functional recovery after TBI(FIG. 25A). However, such improvement was not seen the cylinder test(FIG. 25B), which accesses spontaneous forepaw use in explorativebehavior with a clear ceiling.

Besides a significant improvement in both neurological and motorfunctions observed in the TBI-induced rat model, rats treated withcysteinyl-succinyl crosslinked hemoglobin solution showed significantreduction in TBI-induced astrocyte activation under GFAPimmunofluorescence analysis (*p<0.05; t-test), as depicted in FIG. 26.

Example 23: Cancer Treatment Studies: Tumor Inhibition inTriple-Negative Breast Cancer and Colorectal Cancer

Both triple-negative breast cancer and colorectal cancer are associatedwith poor prognosis and a high mortality rate due to the formation of asolid tumor and an associated hypoxic environment. In searching foreffective therapeutic options, some literature suggests the use ofsystemic oxygenation to inhibit the solid tumor growth and metastasis invarious cancer models, by weakening the hypoxia-A2A adenosine receptors(A2aR)-driven immunosuppression in the tumor microenvironment.Therefore, the cysteinyl-succinyl crosslinked hemoglobin was tested forits role in inhibiting tumor growth in triple-negative breast cancer andcolorectal cancer, respectively.

Example 23A: Inhibition of Tumor Growth in Triple-Negative Breast CancerXenograft Model

A significant inhibition of tumor growth in TNBC 4T1 xenograft wasobserved after administration of cysteinyl-succinyl crosslinkedhemoglobin solution. A murine triple-negative breast carcinoma xenograft(TNBC) model was employed. Murine TNBC 4T1 cells were cultured in DMEMsupplemented with 10% FBS, 4 mM L-glutamine, 1 mM sodium pyruvate, 100U/mL penicillin and 100 ug/mL streptomycin at 37° C. under 5% CO₂.Approximately 1×10⁵ cancer cells (TNBC 4T1 cell line) were injectedsubcutaneously into four to six week-old female immune competent BALB/Cmice. Once the tumor had grown for one week, tumor-bearing mice wererandomized into two groups as follows:

Group 1: Saline Buffer (Control); and

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (400 mgHb/kg of body weight).

For each group, 4-6 mice were given either saline or cysteinyl-succinylcrosslinked hemoglobin solution (400 mg/kg) once weekly for four weeks.Tumor volume was recorded every three days starting with the first dayof treatment. The tumor volume was calculated using the modifiedellipsoidal formula: Tumor Volume=½×LW², where L and W represent thelength and width of the tumor mass, measured by a digital caliper(Mitu-toyo Co, Tokyo, Japan) at each measurement. Results demonstratedthat a suppression of tumor growth (22-51%) in TNBC 4T1 xenograft wasobserved in mice treated with cysteinyl-succinyl crosslinked hemoglobinsolution at 6 days post 2^(nd) injection, compared to the control group,as depicted in Table 18.

TABLE 18 Percentage Change of the Normalized Tumor Volume Against theControl Group for the Syngeneic 4T1 Model. % Change of the NormalizedTumor Volume against the Control Group Experi- Experi- Experi- Experi-Measurement Time ment 1 ment 2 ment 3 ment 4 6 Day Post 1^(st) Injection−39.6% 4.2% −17.0% −25.9% 6 Day Post 2^(nd) Injection −51.2% −23.4%−49.1% −22.4%

Example 23B: Inhibition of Tumor Growth in Colorectal Cancer XenograftModel

A significant inhibition of tumor growth in CT26 xenograft was observedafter administration of cysteinyl-succinyl crosslinked hemoglobinsolution, as shown in Table 13. A murine colorectal carcinoma xenograft(TNBC) model was employed. Murine CT26 cells were cultured in RPMI-1640supplemented with 10% FBS, 100 U/mL penicillin and 100 ug/mLstreptomycin at 37° C. under 5% CO₂. Approximately 1×10¹ cancer cells(CT26 cell line) were injected subcutaneously into four to six week-oldfemale immune competent BALB/C mice. When the tumor grew for one week,tumor-bearing mice were randomized into two groups as follows:

Group 1: Saline Buffer (Control); and

Group 2: Cysteinyl-succinyl Crosslinked Hemoglobin Solution (400 mgHb/kg of body weight)

For each group, 4-6 mice were given either saline or cysteinyl-succinylcrosslinked hemoglobin (400 mg/kg) once weekly for four weeks. Tumorvolume was recorded every three days starting with the first day oftreatment. The tumor volume was calculated using the modifiedellipsoidal formula: Tumor Volume=½×LW², where L and W represent thelength and width of the tumor mass, measured by a digital caliper(Mitu-toyo Co, Tokyo, Japan) at each measurement. Results demonstratedthat a suppression of tumor growth (60%) in CT26 xenograft was observedin mice treated with cysteinyl-succinyl crosslinked hemoglobin solutionat 6 days post 2^(nd) injection, compared to the control group, asdepicted in Table 19.

TABLE 19 Percentage Change of the Normalized Tumor Volume Against theControl Group for the Syngeneic CT26 Model. % Change of the NormalizedTumor Measurement Time Volume against the Control Group 6 Day Post1^(st) Injection −60.77% 6 Day Post 2^(nd) Injection −65.77%

What is claimed is:
 1. A thiosuccinyl-crosslinked hemoglobin comprisinga tetrameric hemoglobin and at least one thiosuccinyl crosslinkingmoiety of Formula 1:

or a pharmaceutically acceptable salt or zwitterion thereof, whereineach N* independently represents a nitrogen selected from the groupconsisting of a nitrogen in a lysine residue side chain in thetetrameric hemoglobin and a nitrogen at a N-terminus in the tetramerichemoglobin; and R¹ is alkyl, alkenyl, cycloalkyl, heterocycloalkyl,aryl, aralkyl, heteroaryl, or —(CR₂)_(n)Y, wherein n is an integerselected from 0-10; R for each instance is independently hydrogen,alkyl, aralkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, orheteroaryl; or two instances of R taken together form a 3-6 memberedcycloalkyl or heterocycloalkyl containing 1, 2, or 3 heteroatomsselected from N, O, and S; and Y is selected from the group consistingof OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴,—(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, —(NR⁴)S(O)₂OR⁴, and—(CRR²R³), wherein R² is hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴,—(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; R³ is hydrogen, alkyl, aralkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, OR⁴, SR⁴, N(R⁴)₂,—(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; and R⁴ for each instance isindependently selected from the group consisting of hydrogen, alkyl,aralkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl; or R¹ is amoiety selected from the group consisting of:

and N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.
 2. The thiosuccinyl-crosslinked hemoglobin ofclaim 1, wherein R¹ is a moiety of Formula 2:

wherein n is a whole number selected from the group consisting of 0, 1,2, 3, and 4; R for each instance is independently selected from thegroup consisting of hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl; R² is hydrogen, alkyl, aralkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, —N(R⁴)₂, —NH(C═O)R⁴, or—NH(C═O)N(R⁴)₂; R³ is hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, —CO₂R⁴, —(C═O)NHR⁴, —OR⁴, or—N(R⁴)₂; and R⁴ for each instance is independently selected from thegroup consisting of hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, and heteroaryl; or R¹ is a moiety selected fromthe group consisting of:

and N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.
 3. The thiosuccinyl-crosslinked hemoglobin ofclaim 2, wherein n is 1 or 2; R is hydrogen; R² is —NHR⁴, —NH(C═O)R⁴, or—NH(C═O)R⁴N(R⁴)₂; and R³ is hydrogen, —OR⁴, —CO₂R⁴, or —(C═O)NHR⁴,wherein R⁴ for each instance is independently selected from the groupconsisting of hydrogen and alkyl.
 4. The thiosuccinyl-crosslinkedhemoglobin of claim 1, wherein R¹ is selected from the group consistingof:

or a pharmaceutically acceptable salt or zwitterion thereof, wherein mis a whole number selected from 1-1000.
 5. The thiosuccinyl-crosslinkedhemoglobin of claim 1, wherein each N* independently represents anitrogen selected from the group consisting of a nitrogen in a lysineresidue side chain in a beta globin chain of the tetrameric hemoglobinand a nitrogen at a N-terminus in a beta globin chain of the tetramerichemoglobin.
 6. The thiosuccinyl-crosslinked hemoglobin of claim 1,wherein the thiosuccinyl-crosslinked hemoglobin is substantially pure.7. The thiosuccinyl-crosslinked hemoglobin of claim 1, wherein thethiosuccinyl-crosslinked hemoglobin comprises 1, 2, or 3 thiosuccinylcrosslinking moiety of Formula
 1. 8. The thiosuccinyl-crosslinkedhemoglobin of claim 1, wherein the at least one thiosuccinylcrosslinking moiety crosslinks two beta globin chains of the tetramerichemoglobin.
 9. The thiosuccinyl-crosslinked hemoglobin of claim 1,wherein the tetrameric hemoglobin is human hemoglobin, bovinehemoglobin, or porcine hemoglobin.
 10. The thiosuccinyl-crosslinkedhemoglobin of claim 1, wherein the thiosuccinyl-crosslinked hemoglobinis substantially stroma-free.
 11. A pharmaceutical compositioncomprising at least one of the thiosuccinyl-crosslinked hemoglobin ofclaim 1 and at least one pharmaceutically acceptable excipient.
 12. Thepharmaceutical composition of claim 11, wherein thethiosuccinyl-crosslinked hemoglobin is present in the pharmaceuticalcomposition at a weight percentage between 10-90%.
 13. Thepharmaceutical composition of claim 11, wherein the pharmaceuticalcomposition comprises thiosuccinyl-crosslinked hemoglobin comprising 1,2, or 3 thiosuccinyl crosslinking moieties of Formula 1; or acombination thereof.
 14. A method for preparing thethiosuccinyl-crosslinked hemoglobin of claim 1 comprising the steps of:contacting a tetrameric hemoglobin with a fumaryl crosslinking agentthereby forming a fumaryl-crosslinked hemoglobin; contacting thefumaryl-crosslinked hemoglobin with a thiol or a pharmaceuticallyacceptable salt or zwitterion thereof thereby forming thethiosuccinyl-crosslinked hemoglobin of claim
 1. 15. The method of claim14, wherein the fumaryl crosslinking agent is selected from the groupconsisting of bis-3,5-dibromosalicyl fumarate (DBSF), fumaryl chlorideand bis(salicyl) fumarate.
 16. The method of claim 14, wherein the thiolhas the formula: R¹SH or a pharmaceutically acceptable salt orzwitterion thereof, wherein R¹ is alkyl, alkenyl, cycloalkyl,heterocycloalkyl, aryl, aralkyl, heteroaryl, or —(CR₂)_(n)Y, wherein nis an integer selected from 0-10; R for each instance is independentlyhydrogen, alkyl, aralkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl,or heteroaryl; or two instances of R taken together form a 3-6 memberedcycloalkyl or heterocycloalkyl containing 1, 2, or 3 heteroatomsselected from N, O, and S; and Y is selected from the group consistingof R¹ is alkyl, alkenyl, cycloalkyl, heterocycloalkyl, aryl, aralkyl,heteroaryl, or —(CR₂)_(n)Y, wherein n is an integer selected from 0-10;R for each instance is independently hydrogen, alkyl, aralkyl, alkenyl,cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; or two instances of Rtaken together form a 3-6 membered cycloalkyl or heterocycloalkylcontaining 1, 2, or 3 heteroatoms selected from N, O, and S; and Y isselected from the group consisting of OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴,—(C═O)OR⁴, —O(C═O)R⁴, —O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴,—(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂, —O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂,—(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂, —(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴,—S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴, —OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂,—(NR⁴)S(O)₂N(R⁴)₂, —(NR⁴)S(O)₂OR⁴, and —(CRR²R³), wherein R² ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴,—O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; R³ ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, OR⁴, SR⁴, N(R⁴)₂, —(C═O)R⁴, —(C═O)OR⁴, —O(C═O)R⁴,—O(C═O)OR⁴, —(C═O)N(R⁴)₂, —(NR⁴)(C═O)R⁴, —(NR⁴)(C═O)OR⁴, —O(C═O)N(R⁴)₂,—O(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═O)N(R⁴)₂, —(C═NR⁴)N(R⁴)₂, —(NR⁴)(C═NR⁴)N(R⁴)₂,—(S═O)R⁴, —S(O)₂R⁴, —S(O)₂OR⁴, —S(O)₂N(R⁴)₂, —OS(O)₂R⁴, —(NR⁴)S(O)₂R⁴,—OS(O)₂OR⁴, —OS(O)₂N(R⁴)₂, —(NR⁴)S(O)₂N(R⁴)₂, or —(NR⁴)S(O)₂OR⁴; and R⁴for each instance is independently selected from the group consisting ofhydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; or R¹ is a moiety selected from the group consisting of:

and N⁵-(1-((carboxymethyl)amino)-1-oxo-3λ³-propan-2-yl)glutamine or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected from 1-1000.
 17. The method of claim 16, wherein the thiol hasthe Formula 3:

or a pharmaceutically acceptable salt or zwitterion thereof, wherein nis an integer selected from the group consisting of 0, 1, 2, 3, and 4; Rfor each instance is independently selected from the group consisting ofhydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; R² is hydrogen, alkyl, aralkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, —N(R⁴)₂, or —NH(C═O)R⁴; R³ ishydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, —CO₂R⁴, —(C═O)NHR⁴, —OR⁴, or —N(R⁴)₂; and R⁴ for eachinstance is independently selected from the group consisting ofhydrogen, alkyl, aralkyl, cycloalkyl, heterocycloalkyl, aryl, andheteroaryl; or the thiol is selected from the group consisting ofdithiothreitol, HS(CH₂CH₂O)_(m)CH₃, HS(CH₂CH₂O)_(m)H, glutathione or apharmaceutically acceptable salt thereof, wherein m is a whole numberselected between 1-1000.
 18. The method of claim 17, wherein n is 1 or2; R is hydrogen; R² is —NHR⁴, —NH(C═O)R⁴, or —NH(C═O)(NR⁴)₂; and R³ ishydrogen, —OR⁴, —CO₂R⁴, or —(C═O)NHR⁴, wherein R⁴ for each instance isindependently selected from the group consisting of hydrogen and alkyl.19. The method of claim 14, wherein the thiol is selected from the groupconsisting of:

dithiothreitol, HS(CH₂CH₂O)_(m)CH₃, and HS(CH₂CH₂O)_(m)H or apharmaceutically acceptable salt or zwitterion thereof, wherein m is awhole number selected between 1-1000.
 20. The method of claim 14,wherein the step of contacting the fumaryl-crosslinked hemoglobin with athiol or a pharmaceutically acceptable salt or zwitterion thereof, thefumaryl-crosslinked hemoglobin and the thiol are present in a molarratio of at least 1:1; 1:2; or 1:3.
 21. The method of claim 20, whereinthe fumaryl-crosslinked hemoglobin and the thiol are present in a molarratio of greater than 1:3.
 22. The method of claim 14, wherein thethiosuccinyl-crosslinked hemoglobin is in isolated and substantiallypure form.
 23. A method for increasing the volume of the bloodcirculatory system in a subject in need thereof, wherein the methodcomprises transfusing into the system of the subject a therapeuticallyeffective amount of the thiosuccinyl-crosslinked hemoglobin of claim 1.24. A method for the treatment of shock in a subject in need thereof,wherein the method comprises transfusing into the system of the subjecta therapeutically effective amount of the thiosuccinyl-crosslinkedhemoglobin of claim
 1. 25. A method of supplying oxygen to the tissuesand organs in a subject in need thereof, wherein the method comprisestransfusing into the system of the subject a therapeutically effectiveamount of the thiosuccinyl-crosslinked hemoglobin of claim
 1. 26. Amethod of treating cancer in a subject in need thereof, wherein themethod comprises transfusing into the system of the subject atherapeutically effective amount of the thiosuccinyl-crosslinkedhemoglobin of 1, wherein the cancer is triple-negative breast cancer orcolorectal cancer.
 27. The method of claim 22, whereinthiosuccinyl-crosslinked hemoglobin is substantially pure.